Factors for the production and accumulation of polyunsaturated fatty acids (pufas) derived from pufa synthases

ABSTRACT

Factors For the Production and Accumulation of Polyunsaturated Fatty Acids (PUFAs) Derived from PUFA Synthases Abstract Disclosed are novel enhancing factor proteins of the PUFA synthase systems, nucleic acid molecules encoding the same, recombinant nucleic acid molecules and recombinant host cells comprising such nucleic acid molecules, genetically modified microorganisms comprising the same, and methods of making and using the same. Also disclosed are genetically modified microorganisms that have been genetically modified to express a PUFA synthase system for the production of PUFAs, wherein the microorganisms have been modified to express the novel enhancing factor proteins of the PUFA synthase system.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the filing date of U.S.Provisional Patent Application No. 61/932,310 filed Jan. 28, 2014, thedisclosure of which is hereby incorporated herein by reference.

FIELD OF THE INVENTION

The present invention generally relates to the identification and use ofenhancing factor proteins to improve the production of polyunsaturatedfatty acids (PUFAs) and particularly, long chain PUFAs (LCPUFAs), in ahost organism that has been genetically modified with a PUFA synthasesystem for producing such PUFAs. The present invention also relates tothe organisms that have been genetically modified to express suchenhancing factor proteins or modified with respect to such proteins, andto methods of making and using such microorganisms.

BACKGROUND OF THE INVENTION

Polyunsaturated fatty acids (PUFAs) are considered to be useful fornutritional applications, pharmaceutical applications, industrialapplications, and other purposes. However, the current supply of PUFAsfrom natural sources and from chemical synthesis is not sufficient forcommercial needs. PUFAs derived from microorganism such as microalgaecan be produced in large scale while avoiding the contamination issuesassociated with fish oils.

Polyketide synthase (PKS) systems are generally known in the art asenzyme complexes related to fatty acid synthase (FAS) systems, but whichare often highly modified to produce specialized products that typicallyshow little resemblance to fatty acids. It has now been shown, however,that polyketide synthase-like systems exist in marine bacteria andcertain microalgae that are capable of synthesizing polyunsaturatedfatty acids (PUFAs) from acetyl-CoA and malonyl-CoA. These systems arereferred to herein as PUFA synthases, PUFA synthase systems, PUFA PKSsystems, or PKS-like systems for the production of PUFAs, all of whichare used interchangeably herein.

The PUFA PKS pathways for PUFA synthesis in Shewanella and anothermarine bacteria, Vibrio marinus, are described in detail in U.S. Pat.No. 6,140,486. The PUFA PKS pathways for PUFA synthesis in theeukaryotic Thraustochytrid, Schizochytrium sp. ATCC 20888 (hereinafter“Schizochytrium 20888”), is described in detail in U.S. Pat. No.6,566,583. The PUFA PKS pathways for PUFA synthesis in eukaryotes suchas members of Thraustochytriales, including the additional descriptionof a PUFA PKS system in Schizochytrium 20888 and the identification of aPUFA PKS system in Thraustochytrium sp. ATCC 20892, including detailsregarding uses of these systems, are described in detail in U.S. Pat.No. 7,247,461, and in U.S. Pat. No. 7,642,074, respectively. The PUFAPKS pathways for PUFA synthesis in another eukaryotic Thraustochytrid,Schizochytrium sp. ATCC PTA-9695 (hereinafter “Schizochytrium 9695”), isdescribed in detail in U.S. Patent Application Publication No.2010-0266564, published Oct. 21, 2010 and in PCT Publication No. WO2010/108114, published Mar. 19, 2010. U.S. Pat. No. 7,211,418, disclosesthe detailed structural description of a PUFA PKS system inThraustochytrium sp. ATCC 20892, and further detail regarding theproduction of eicosapentaenoic acid (C20:5, n-3) (EPA) and other PUFAsusing such systems. U.S. Pat. No. 7,217,856 discloses the structural andfunctional description of PUFA PKS systems in Shewanella olleyana andShewanella japonica, and uses of such systems. These applications alsodisclose the genetic modification of organisms with the genes comprisingthe PUFA PKS pathway and the production of PUFAs by such organisms.Furthermore, U.S. Pat. No. 7,776,626 describes a PUFA PKS system inUlkenia, and U.S. Pat. No. 7,208,590 describes PUFA PKS genes andproteins from Thraustochytrium aureum. Each of the above-identifiedapplications is incorporated by reference herein in its entirety.

Accordingly, the basic domain structures and sequence characteristics ofthe PUFA synthase family of enzymes have been described, and it has beendemonstrated that PUFA synthase enzymes are capable of de novo synthesisof various PUFAs (e.g., eicosapentaenoic acid (EPA; C20:5, n-3),docosahexaenoic acid (DHA; C22:6, n-3) and docosapentaenoic acid(DPAn-6; C22:5, n-6).

PUFA synthases produce long chain polyunsaturated fatty acids de novofrom malonyl-CoA using NADPH (and perhaps NADH) as a reductant. Thesemulti-subunit enzymes have been identified in both marine bacteria andin the eukaryotic Thraustochytrid group of marine algae (Metz et al.,2001, Science 293:290-293). All of the PUFA synthases identified to datecontain multiple ACP domains upon which the fatty acids are assembled.ACP domains require attachment of a co-factor by a phosphopantetheinyltransferase (PPTase) in order to function. Individual PPTases can haveACP substrate preferences, and when expressing a PUFA synthase in aheterologous organism, it may be necessary to provide a PPTase that canrecognize, and activate, its ACP domains.

Novel production of PUFAs in several heterologous host organisms hasbeen achieved by expression of the genes encoding the PUFA synthasesubunits along with an appropriate PPTase. Of particular interest here,is the PUFA synthase derived from Schizochytrium 20888 (Metz et al.,2001, Science 293:290-293). The primary products of this PUFA synthaseare DHA and DPAn-6. Schizochytrium 20888 has been developed as acommercial source for oil enriched in DHA. The organism can accumulatehigh levels of oil (>60% of the biomass) and DHA can comprise >40% ofthe fatty acids present in that biomass (Barclay et al., Single CellOils, 2^(nd) edition. 2010 AOCS Press, pgs 75-96). In the nativeorganism the DHA to DPAn-6 ratio typically ranges between 2.3 to 2.7.Expression of the PUFA synthase subunits of Schizochytrium 20888, alongwith an appropriate PPTase (e.g., HetI from Nostoc sp., see Hauvermaleet al., 2008, Lipids 41: 739-747 and Metz et al., US Patent ApplicationPublication No. 2013-0150599) in heterologous host cells has resulted inproduction of DHA and DPAn-6 in those cells. Although DHA and DPAn-6 areproduced in cells of E. coli (Hauvermale et al., 2008, Lipids41:739-747), and in yeast and higher plants (Metz et al., US PatentApplication Publication No. 2013-0150599), the levels have notapproached those observed in the native organism. Additionally, in bothyeast and in plants, the DHA to DPAn-6 ratio is typically significantlylower than that observed in the native organism. It is possible thatsome factor (or factors) in addition to the activated subunits of theenzyme itself is present in the cells of Schizochytrium 20888 whichfacilitates activity of the enzyme.

Since additional factors involved in the PUFA synthesis mechanism canhave implications for increasing the efficiency of and/or improving theproduction of PUFAs in an organism that has been genetically modified toproduce such PUFAs, there is a need in the art for finding suchfactor/factors. Accordingly, there is also a need in the art forimproved methods of production of PUFAs, including in microorganismsthat have been genetically modified to produce such PUFAs, which takeadvantage of the activity of such mechanism.

SUMMARY OF THE INVENTION

One embodiment of the invention relates to a recombinant nucleic acidmolecule comprising a nucleic acid sequence encoding a polypeptide thatis at least 85% identical to an amino acid sequence of SEQ ID NO:1 orSEQ ID NO:3.

In one aspect, the recombinant nucleic acid molecule comprises a nucleicacid sequence encoding a polypeptide that is at least 90% identical toan amino acid sequence of SEQ ID NO:1 or SEQ ID NO:3. In another aspect,the recombinant nucleic acid molecule comprises a nucleic acid sequenceencoding a polypeptide that is at least 95% identical to an amino acidsequence of SEQ ID NO:1 or SEQ ID NO:3. In one aspect, the polypeptideenhances the enzymatic activity of a PUFA synthase. In one aspect, therecombinant nucleic acid molecule comprises a nucleic acid sequenceencoding a polypeptide that is an enzymatically active fragment of SEQID NO:1 or SEQ ID NO:3. In one aspect, the nucleic acid sequence encodesa polypeptide having an amino acid sequence of SEQ ID NO:1 or SEQ IDNO:3. In another aspect, the nucleic acid sequence is SEQ ID NO:5 or SEQID NO:7.

Another embodiment of the invention relates to a recombinant nucleicacid molecule comprising a nucleic acid sequence encoding a polypeptidethat is at least 85% identical to an amino acid sequence of SEQ ID NO:2or SEQ ID NO:4.

In one aspect, the recombinant nucleic acid molecule comprises a nucleicacid sequence encoding a polypeptide that is at least 90% identical toan amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4. In another aspect,the recombinant nucleic acid molecule comprises a nucleic acid sequenceencoding a polypeptide that is at least 95% identical to an amino acidsequence of SEQ ID NO:2 or SEQ ID NO:4. In one aspect, the polypeptideenhances the enzymatic activity of a PUFA synthase. In one aspect, therecombinant nucleic acid molecule comprises a nucleic acid sequenceencoding a polypeptide that is an enzymatically active fragment of SEQID NO:2 or SEQ ID NO:4. In one aspect, the nucleic acid sequence encodesa polypeptide having an amino acid sequence of SEQ ID NO:2 or SEQ IDNO:4. In another aspect, the nucleic acid sequence is SEQ ID NO:6 or SEQID NO:8.

Another embodiment of the invention relates to an isolated proteinencoded by any of the above-described nucleic acid molecules.

Another embodiment of the invention relates to a recombinant nucleicacid molecule comprising any of the above-described nucleic acidmolecules, which is operatively linked to an expression controlsequence.

Yet another embodiment of the invention relates to a recombinant hostcell comprising any of the above-described nucleic acid molecules. Inone aspect, this recombinant host cell is a microorganism.

Another embodiment of the invention relates to a genetically modifiedmicroorganism, wherein the microorganism has been genetically modifiedto express any one of the above-described nucleic acid molecules.

In another embodiment, the microorganism has been genetically modifiedto express one of the above-described recombinant nucleic acid moleculesderived from SEQ ID NO:5 or SEQ ID NO:7, and another recombinant nucleicacid molecule derived from SEQ ID NO:6 or SEQ ID NO:8.

In one embodiment, the microorganism endogenously expresses a PUFAsynthase system, a phosphopantetheinyl transferase (PPTase), and/or anacyl-CoA synthetase (ACS). In one aspect, the microorganism is aThraustochytriales microorganism. In one aspect, the microorganism is aSchizochytrium. In one aspect, the microorganism is a bacterium.

In another embodiment, the microorganism has been genetically modifiedto exogenously express a PUFA synthase system, a phosphopantetheinyltransferase (PPTase), and/or an acyl-CoA synthetase (ACS). In oneaspect, the microorganism is a Thraustochytriales microorganism. In oneaspect, the microorganism is a Schizochytrium. In one aspect, themicroorganism is a microalga. In one aspect, the microorganism is ayeast. In one aspect, the microorganism is a bacterium. In one aspect,the PUFA synthase comprises at least one functional domain from a PUFAsynthase from a microorganism selected from the group consisting ofSchizochytrium sp. American Type Culture Collection (ATCC) No. 20888,Schizochytrium sp. American Type Culture Collection (ATCC) No. PTA-9695,Thraustochytrium 23B American Type Culture Collection (ATCC) No. 20892,and a mutant of any of said microorganisms. In one aspect, the PUFAsynthase comprises at least one functional domain from a PUFA synthasefrom a marine bacterium.

In one aspect, the one or more nucleic acid sequences encoding the PUFAsynthase of the above-described genetically modified microorganism hasbeen optimized to improve the expression of the PUFA synthase in themicroorganism. In one aspect, the genetically modified microorganismcomprises at least one polyunsaturated fatty acid (PUFA) selected fromthe group consisting of: DHA (C22:6, n-3), DPA (C22:5, n-6 or n-3), EPA(C20:5, n-3), ARA (C20:4, n-6), GLA (C18:3, n-6), and/or SDA (C18:4,n-3). In a preferred aspect, the genetically modified microorganismcomprises DHA, DPAn-6 and/or EPA.

In one aspect, the amount of DHA, DPAn-6 and/or EPA produced in theabove-described genetically modified microorganism, wherein suchmicroorganism is modified to express at least two of the above-describedrecombinant nucleic acid molecules,—one comprises a nucleic acidsequence encoding a polypeptide that is at least 90% identical to anamino acid sequence of SEQ ID NO:1 or SEQ ID NO:3, and another comprisesa nucleic acid sequence encoding a polypeptide that is at least 90%identical to an amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4, ishigher than that is produced in the counterpart microorganism which noneof the above-described recombinant nucleic acid molecule is expressed.

In one aspect, the ratio of DHA:DPAn-6 produced in the above-describedgenetically modified microorganism, wherein such microorganism ismodified to express at least two of the above-described recombinantnucleic acid molecules,—one comprises a nucleic acid sequence encoding apolypeptide that is at least 90% identical to an amino acid sequence ofSEQ ID NO:1 or SEQ ID NO:3, and another comprises a nucleic acidsequence encoding a polypeptide that is at least 90% identical to anamino acid sequence of SEQ ID NO:2 or SEQ ID NO:4, is higher than thatis produced in the counterpart microorganism which none of theabove-described recombinant nucleic acid molecule is expressed.

In one aspect, the microorganism is a microalga, a yeast, or abacterium.

In another embodiment, the invention provides a genetically modifiedmicroorganism, wherein the microorganism has been genetically modifiedto delete or inactivate one of the above-described recombinant nucleicacid molecules expressed by the microorganism. In one aspect, themicroorganism is a Thraustochytriales microorganism. In one aspect, themicroorganism is a Schizochytrium.

In one embodiment, the invention provides an oil obtained from one ofthe above-described genetically modified microorganism.

In one embodiment, the invention provides a method to produce an oilcomprising at least one polyunsaturated fatty acid (PUFA), comprisinggrowing one of the above-described genetically modified microorganism.

In one embodiment, the invention provides an oil produced by theabove-described method. In one aspect, the oil contains at least onepolyunsaturated fatty acid (PUFA) selected from the group consisting of:DHA (C22:6, n-3), DPA (C22:5, n-6 or n-3), and/or EPA (C20:5, n-3).

In one embodiment, the invention provides a food product or feed productthat contains the above-described oil or genetically modifiedmicroorganisms.

In one embodiment, the invention provides a pharmaceutical product thatcontains the above-described oil.

In one embodiment, the invention provides a method to produce an oilcomprising at least one PUFA, comprising recovering an oil from one ofthe above-described genetically modified microorganisms.

In one embodiment, the invention provides a method to produce at leastone polyunsaturated fatty acid (PUFA), comprising growing theabove-described genetically modified microorganisms.

In one embodiment, the invention provides a method to produce at leastone polyunsaturated fatty acid (PUFA), comprising obtaining orrecovering the PUFA from one of the above-described genetically modifiedmicroorganisms.

In one embodiment, the invention provides a method to provide asupplement or therapeutic product containing at least one PUFA to anindividual, comprising providing to the individual the above-describedgenetically modified microorganisms, oil, food products, orpharmaceutical products.

In one embodiment, the invention provides a process for transforming amicroorganism to express PUFAs, comprising transforming a microorganismwith nucleic acid molecules encoding a PUFA synthase, with a nucleicacid molecule encoding a phosphopantetheinyl transferase (PPTase), witha nucleic acid molecule encoding an acyl-CoA synthetase (ACS), and withat least one of the above-described nucleic acid molecules.

BRIEF DESCRIPTION OF THE DRAWINGS OF THE INVENTION

FIG. 1 is a diagram showing the result of an in vitro PUFA synthaseactivity enhancing assay using enhancing factor proteins Sz-TE2 andSz-TE3 expressed in E. coli.

FIG. 2 is a diagram showing the result of an in vitro PUFA synthaseactivity enhancing assay using extracts from the Schizochytrium 20888Quad-KO strain or extracts from cells derived from that strain in whicheither Sz-TE2 or Sz-TE3 have been inactivated.

FIG. 3A is a diagram showing the alignment between the amino acidsequence of enhancing factor proteins B-TE2 and Sz-TE2.

FIG. 3B is a diagram showing the alignment between the amino acidsequence of enhancing factor proteins B-TE3 and Sz-TE3.

FIG. 4 is a diagram showing an in vitro PUFA synthase activity enhancingassay using separately expressed B-TE2 and B-TE3.

SEQUENCE LISTING

The nucleic acid sequences and deduced amino acid translation sequenceslisted in the accompanying sequence listing are shown using standardletter abbreviations for nucleotide bases and amino acids, as defined in37 C.F.R. §1.822. Only one strand of each nucleic acid sequence isshown, but the complementary strand is understood to be included by anyreference to the displayed strand. In the accompanying sequence listing:

SEQ ID NO:1 shows the amino acid of the TE2 protein from Schizochytriumsp. ATCC-20888 (Sz-TE2):

  1 MTAQGGYRSE MLMYYEDTDL TGAVYAGNYF KYFERARDEA VGIDVLKTLM DKEGLALYVR 61 KMGEMTFKGG AKHADTLVVE SSVEAPSDFR LVFKQRASVK DRPETIIVET DVEVVCIDMK121 TQRVAKIPTQ IREALRI

SEQ ID NO:2 shows the amino acid sequence of the TE3 protein fromSchizochytrium sp. ATCC 20888 (Sz-TE3):

  1 MAAPSTAVCG ELPKLDEAPL KVSRARGYDA LDVKVYREDT DTVGIVFYRN FLTWFERGRE 61 NAISTDFLAG LFEYSGDSFV VTRSEQSFRK PAFYGDELEV RTIPFADGPF RLHFDQSIWR121 KSDNTLLVAG FVEMVTVSRT FQLTKVPQPV HDLIYYFDDC KSNFTYCEKP GAKKRPLRRK181 PGAPSSLGKT TELDLVIHLA DTDFTGIAFH PNYYCWFERA RSDFLSNEIL ARAKTEFHAV241 PVVRSAKLAY KNGARPVEPL RITTTQDPKG EHSDFVVPIL QKLTRVSNDQ TLVEAVFEMC301 FVHDKERHLV KVPSIVRDAI A

SEQ ID NO:3 shows the amino acid sequence of the TE2 protein fromSchizochytrium sp. ATCC PTA-9695 (B-TE2):

  1 MVMVAEEKRA HEVAVQLYYE DTDFSGFVHH ANFLRYFERG RDEMIGLPVL KCLAQDDSSS 61 SSSATSIGGG EPPVSLFVHK VHELSFKGRA RHGEMLVVRS RVVKESDFRL RFAHEAWVGN121 TLVASGSMDV VFLCGSVDAR LVKIPNSVDV ALHGYY

SEQ ID NO:4 shows the amino acid sequence of the TE3 protein fromSchizochytrium sp. ATCC PTA-9695 (B-TE3):

  1 MRIDEEAIRV AAARGYDALP VTVYREFTDC LGIVFYRHYL AWFERGRENV ISVQFLADLF 61 RETGESFVVT RSEQVFKRSA RYGDQLEVRT IPFLDGDYRL GFDQSVWHGN EMLVHGFVEM121 VCVSKSFQLA QQPALVRKLI GCFDECTRNF TYVGTKARMP QTIRRRRGTA SLPQAQKPLV181 FDGLHLHQAD TDFTGITFHP NYYCYFERAR SQALTPAVLA NVAEFANAVP VIRQARMTFK241 QGARAYETLR VLTSIALDDS GGSSSSSNKY VVPFEQVLVR REDDKVLVEA RIEIVFVDQT301 TKLPCPIPDA VAAKMQELFA V

SEQ ID NO:5 shows the nucleotide sequence encoding the TE2 protein fromSchizochytrium sp. ATCC 20888 (SEQ ID NO:1):

  1 atgacggcgc agggcggcta cagatcggag atgctcatgt actatgagga cacggacctg 61 accggagccg tctatgcggg caactacttc aagtactttg agcgcgcgcg cgacgaggct121 gtgggcatcg atgtcctcaa gacgctcatg gacaaggagg gcctggcttt gtacgtgcgc181 aaaatgggcg agatgacctt taaaggaggc gccaagcacg ccgacacgct cgtcgtcgag241 tcctctgtcg aggctccctc ggactttcgc cttgtgttca agcagcgggc atccgtcaag301 gaccgtcccg agacgatcat tgtcgagacc gatgttgagg tcgtttgcat cgacatgaaa361 acgcagcgtg tcgccaagat cccgacgcaa atccgggaag cacttcgtat c

SEQ ID NO:6 shows the nucleotide sequence encoding the TE3 protein fromSchizochytrium sp. ATCC 20888 (SEQ ID NO:2):

  1 atggctgcgc catcgactgc agtctgcggc gagctgccaa agctcgacga ggcgcctctc 61 aaggtgtctc gtgcacgtgg ctacgacgcg ctcgacgtca aggtgtacag agaggacaca121 gacacagtag ggatcgtgtt ctatcgtaac tttttgacct ggtttgagcg tggccgggaa181 aacgcgatct ccacagactt tctcgcagga ctgttcgagt acagtggtga ctccttcgtg241 gtcacgcggt ccgagcagtc gtttcgcaag cctgcatttt acggcgatga actcgaagtc301 cgaaccattc cttttgcaga tgggcccttt cgcctgcact ttgaccagag catctggcga361 aagagcgaca acacattgct agtcgctggc tttgtagaga tggtcacggt gagcagaact421 tttcagctca ccaaggtacc tcagccggtg cacgacctca tttattactt tgacgattgc481 aagtcgaact tcacctactg cgaaaagccc ggcgccaaga aaaggccgct tcggcgtaag541 cccggggcgc cctcttcact tggcaaaacc acagagcttg acctggtcat tcacttggcc601 gacactgact ttactggaat cgcattccac cccaactact actgttggtt cgagcgtgcg661 cgctcggatt ttctcagcaa tgagattctt gcacgcgcca agaccgagtt tcatgctgtt721 cccgttgtgc gcagtgcaaa actcgcgtac aaaaacggcg cgaggcctgt tgagccgctc781 cgcattacaa cgacgcaaga tccgaagggc gagcactcgg actttgtcgt accgattctt841 caaaagctta cgcgtgtctc gaacgaccag acgctcgtcg aagccgtctt tgagatgtgc901 tttgttcatg acaaggagcg ccacctcgtc aaggtcccgt cgatcgttcg cgatgctatt961 gcg

SEQ ID NO:7 shows the nucleotide sequence encoding the TE2 protein fromSchizochytrium sp. ATCC PTA-9695 (SEQ ID NO:3):

  1 atggtcatgg tcgcggagga gaagagggcg cacgaggtgg cagtacagtt gtactatgag 61 gacacggact tctccggctt tgtccatcat gccaacttcc tgcgctactt tgaacgcggc121 cgggatgaga tgattggcct gcccgttctc aaatgcttgg cccaagacga tagctcttct181 tcttcttctg caacttcaat tggtggtggc gagcctccag tatcattgtt cgtgcataag241 gtgcacgagt tgtcgttcaa aggtcgcgct cggcacggtg agatgctcgt ggtgcggtca301 cgagtggtca aggaatcgga cttccgactg cgctttgcac acgaagcgtg ggtggggaac361 acgctcgtgg cctctggatc aatggacgtg gtgttcctgt gtggctcggt cgatgcgcga421 ttagtgaaga tccctaactc ggtcgatgtg gccttgcacg gatactat

SEQ ID NO:8 shows the nucleotide sequence encoding the TE3 protein fromSchizochytrium sp. ATCC PTA-9695 (SEQ ID NO:4):

  1 atgagaatcg acgaggaggc gatacgcgtg gcagcggcgc gcgggtacga cgccttgccc 61 gtgacagtgt atcgagagtt taccgactgc ctgggcattg tgttctaccg gcactaccta121 gcgtggtttg agcgcgggcg cgagaacgtc atctcggtgc agttcttggc ggatctgttt181 cgcgaaacgg gggagtcgtt cgtggtgacg cgctccgagc aagtgtttaa gcgctcagcg241 cgctatggcg accaactcga agtgcgcacc attcctttcc tggacggcga ctaccgcctc301 ggcttcgacc agagcgtgtg gcacggcaat gagatgctcg tgcatggctt cgtggagatg361 gtctgcgtaa gcaagagctt ccagctggcg caacaaccgg cgctcgtgcg caagctgatc421 ggctgctttg acgagtgcac gcgcaacttc acctacgtcg gcaccaaggc ccgcatgccc481 caaaccattc gacgacgcag aggcacggcc agtctaccac aagcacagaa gcctctagtg541 tttgacgggc tgcacttgca ccaagcggac acagacttca caggtatcac ttttcacccc601 aactactact gctactttga acgcgcgcgc tcgcaggcat tgactcccgc cgtattagcg661 aacgtggctg agttcgccaa cgctgtgcca gtcatccgcc aagcccgcat gaccttcaag721 caaggcgcga gagcgtacga gacactccgc gtgctcacat caattgctct ggatgatagc781 ggcggcagca gcagcagcag caacaagtat gtcgtgccgt ttgagcaggt gctcgtgcga841 agagaagacg acaaggtgct ggtggaggcg cgaatcgaga ttgtctttgt ggaccagact901 acgaagttgc cctgcccgat tcctgacgca gtggcagcca agatgcagga gttgtttgcg961 gta

DETAILED DESCRIPTION OF THE INVENTION Abbreviations

ACS acyl-CoA synthetase

B-TE2 or B_TE2 PUFA synthase activity enhancing factor 2 protein fromSchizochytrium 9695

B-TE3 or B_TE3 PUFA synthase activity enhancing factor 3 protein fromSchizochytrium 9695

DHA docosahexaenoic acid

DPAn-6 docosapentaenoic acid n-6

EF-X PUFA synthase in vitro activity enhancing factor proteins

EPA eicosapentaenoic acid

FAME fatty acid methyl ester

FAS fatty acid synthase

HPLC high-performance liquid chromatography

KO knock-out

LCPUFA long chain polyunsaturated fatty acid

PKS polyketide synthase

PPTase phosphopantetheinyl transferase

PUFA polyunsaturated fatty acid

Sz-TE2 or SzTE2 PUFA synthase activity enhancing factor 2 protein fromSchizochytrium 20888

Sz-TE3 or SzTE3 PUFA synthase activity enhancing factor 3 protein fromSchizochytrium 20888

Schizochytrium 9695 Schizochytrium sp. strain ATCC PTA-9695

Schizochytrium 20888 Schizochytrium sp. strain ATCC 20888

The present invention generally relates to the provision of proteins(generally referred to herein as “PUFA synthase in vitro activityenhancing factor proteins” or “EF-X”), and nucleic acid moleculesencoding such proteins, for the improvement of the production ofpolyunsaturated fatty acids (PUFAs) and particularly, long chain PUFAs(LCPUFAs), in a host organism that has been genetically modified toproduce such PUFAs. The present invention also relates to the organismsthat have been genetically modified to express certain of such proteins,and to methods of making and using such proteins and organisms.

According to the present invention, an organism that has beengenetically modified to express a PUFA synthase system, wherein theorganism does not naturally (endogenously, without genetic modification)express such a system, can be referred to herein as a “heterologous”host organism with regard to the modification of the organism with thePUFA synthase system. The genetic modifications of the present inventionmay also be used to improve PUFA production in a host organism thatendogenously expresses a PUFA synthase system, where the organism is notfurther modified with a different PUFA synthase system or a portionthereof, but is genetically modified to express the enhancing factorproteins described herein.

More particularly, the present inventors have discovered and disclosefor the first time a class of enhancing factor proteins which enhancethe activity of PUFA synthases. The present inventors have determinedthat an endogenous producer of PUFA by the PUFA synthase system, i.e.,Schizochytrium 20888, possesses one or more enhancing factor proteinsthat may be capable to enhance the production of PUFAs. This is evidentby the fact that the expression of these factors, especiallyco-expression of the two of such enhancing factor proteins inheterologous host organisms that are also expressing an active PUFAsynthase system from Schizochytrium 20888 results in increasedaccumulation of PUFAs and increased ratio of DHA:DPAn-6 in the cells ofthose organisms. Disruption of the endogenous genes encoding theseproteins in Schizochytrium 20888 leads to a decrease in accumulation ofPUFAs in those cells.

The present inventors have identified from Schizochytrium 20888 twoenhancing factor proteins: SEQ ID NO:1 and SEQ ID NO:2. They are notpart of the PUFA synthase system itself but were found to enhance the invitro activity of PUFA synthases. Both of the enhancing factor proteinshave homology to a class of thioesterases called4-hydroxybenzoyl-CoA-like thioesterases. However, the enhancing factorproteins may or may not have thioesterase activity. SEQ ID NO:1(referred to herein as the enhancing factor 2 protein fromSchizochytrium 20888, or Sz-TE2 protein) has one thioesterase domain,while SEQ ID NO:2 (referred to herein as the enhancing factor 3 proteinfrom Schizochytrium 20888, or Sz-TE3 protein) has two thioesterasedomains. The Sz-TE2 protein and the Sz-TE3 protein function mostefficiently when they are expressed together. See, for example, Example4.

Homologs of these two proteins have also been identified in anotherThraustochytrid-Schizochytrium sp. strain ATCC PTA-9695 which isreferred to here as ‘Schizochytrium 9695’. SEQ ID NO:3 is referred toherein as the enhancing factor 2 protein from Schizochytrium 9695(referred to herein as B-TE2 protein). SEQ ID NO:4 is referred to hereinas the enhancing factor 3 protein from Schizochytrium 9695 (referred toherein as B-TE3 protein). These homologs were shown in the presentinvention to have in vitro PUFA synthase enhancing activity that issimilar to the homolog proteins from Schizochytrium 20888. See, forexample, Examples 9 and 10.

Sz-TE2 and B-TE2 are referred to herein in general as TE2 enhancingfactor proteins. Sz-TE3 and B-TE3 are referred to herein in general asTE3 enhancing factor proteins. It was discovered in the presentinvention that TE2, TE3, and in particular, the combination of TE2 andTE3, enhance the enzymatic activity of PUFA synthase in a host cellwhere TE2 and TE3 are exogenously expressed.

Thioesterases are a class of enzymes which have been extensively studiedin the past. A polypeptide or a domain of a polypeptide havingthioesterase activity has been previously shown to be capable ofcatalyzing the hydrolysis of the thioester bond of fatty acids bound toCoA or acyl carrier proteins. Members of thioesterase enzymes have beenclassified into families by primary structure and into clans andsuperfamilies by tertiary structure (see Cantu et al., 2010, ProteinScience, 19:1281-1295). More than eighty crystal structures ofthioesterases or their domains are available, which provide a wealth ofinformation on the tertiary structures, catalytic residues, andmechanisms of the enzymes. ibid. Some thioesterases have the feature ofa hot dog fold (see Pidugu et al., 2009, BMC Structural Biology, 9:37ppl-16). The hotdog fold was first identified in the crystal structureof β-hydroxydecanoyl thioester dehydratase (Fab A) from E. coli (seeLeesong et al., 1996, Structure 4(3):253-264). According to Leesong etal., at least eight types of hotdog fold thioesterases have beenidentified and their crystal structures were made available, including4-hydroxybenzoyl-CoA thioesterase. Among the subfamilies ofthioesterase, the subfamily of 4-hydroxybenzoyl-CoA thioesterases sharesthe closest protein sequence homology to the TE2 and TE3 proteinsidentified in the present invention.

The present inventors believe that the enhancing factor proteinsdiscovered by the present inventors are useful for modifying PUFAaccumulation in hosts expressing a PUFA synthase, i.e., increasing ordecreasing the amount of the PUFA accumulation and/or changing the ratioof the PUFA products. Indeed, the Examples presented herein demonstratethat the enhancing factor proteins from Schizochytrium 20888 increasethe accumulation of PUFAs in those E. coli and yeast strains which havebeen genetically modified with a Schizochytrium 20888 PUFA synthasesystem. In addition, the enhancing factor proteins alter the ratio ofthe PUFA products of the PUFA synthase system. Each of these enhancingfactor proteins and the nucleic acids encoding the same are encompassedby the present invention, as well as homologues and biologically activefragments thereof. These proteins and nucleic acid molecules will bediscussed in detail below and in the Examples.

One embodiment of the present invention relates to isolated enhancingfactor proteins that enhance the amount and alter the ratio of theproducts of a PUFA synthase system. In one aspect of the invention, theisolated enhancing factor protein(s) is (are) derived from an organismthat endogenously expresses a PUFA synthase system. Such organismsinclude, but are not limited to, members of the Thraustochytriales. Inone aspect, the isolated proteins are derived from the genusSchizochytrium. In another aspect, the isolated enhancing factor proteinis derived from Schizochytrium 20888 or from Schizochytrium 9695. Inanother aspect, any protein that is a homolog of the enhancing factorproteins identified in the present invention and that functions inconjunction with any PUFA synthase system to modify the productionand/or accumulation of PUFAs in a host cell or organism can be used inthe present invention. The invention is not limited to those specificexamples described herein.

In another aspect, the isolated enhancing factor protein is encoded by anucleotide sequence selected from any one of SEQ ID NOs: 5, 6, 7, or 8.In another aspect, the isolated enhancing factor protein is encoded by adegenerate nucleic acid sequence encoding a protein that is encoded by anucleotide sequence selected from any one of SEQ ID NOs: 5, 6, 7, or 8.SEQ ID NO:5 is the nucleotide sequence encoding the TE2 protein fromSchizochytrium 20888. SEQ ID NO:6 is the nucleotide sequence encodingthe TE3 protein from Schizochytrium 20888. SEQ ID NO:7 is the nucleotidesequence encoding the TE2 protein from Schizochytrium 9695. SEQ ID NO:8is the nucleotide sequence encoding the TE3 protein from Schizochytrium9695.

In yet another aspect, the isolated enhancing factor protein comprisesan amino acid sequence selected from any one of SEQ ID NOs:1, 2, 3, 4,or a homologue of any of such amino acid sequences, including anybiologically active fragments or domains of such sequences.

In some embodiment, an enhancing factor protein of the present inventionincludes, for example and without limitation, at least one proteincomprising an amino acid sequence having at least 50% (e.g., at least55%; at least 60%; at least 65%; at least 70%; at least 75%; at least80%; at least 81%; at least 82%; at least 83%; at least 84%; at least85%; at least 86%; at least 87%; at least 88%; at least 89%; at least90%; at least 91%; at least 92%; at least 93%; at least 94%; at least95%; at least 96%; at least 97%; at least 98%; at least 99%) identity tothe amino acid sequence of SEQ ID NO:1 or SEQ ID NO:3.

In some embodiment, an enhancing factor protein includes, for exampleand without limitation, at least one protein comprising an amino acidsequence having at least 50% (e.g., at least 55%; at least 60%; at least65%; at least 70%; at least 75%; at least 80%; at least 81%; at least82%; at least 83%; at least 84%; at least 85%; at least 86%; at least87%; at least 88%; at least 89%; at least 90%; at least 91%; at least92%; at least 93%; at least 94%; at least 95%; at least 96%; at least97%; at least 98%; at least 99%) identity to the amino acid sequence ofSEQ ID NO:2 or SEQ ID NO:4.

One embodiment of the invention relates to an isolated nucleic acidmolecule comprising a nucleic acid sequence encoding a polypeptide thatis at least 50% (e.g., at least 55%; at least 60%; at least 65%; atleast 70%; at least 75%; at least 80%; at least 81%; at least 82%; atleast 83%; at least 84%; at least 85%; at least 86%; at least 87%; atleast 88%; at least 89%; at least 90%; at least 91%; at least 92%; atleast 93%; at least 94%; at least 95%; at least 96%; at least 97%; atleast 98%; at least 99%) identity to the amino acid sequence of SEQ IDNO:1 or SEQ ID NO:3. In one aspect, the polypeptide enhances theenzymatic activity of a PUFA synthase. In one aspect, the isolatednucleic acid molecule comprising a nucleic acid sequence encodes apolypeptide having an amino acid sequence of SEQ ID NO:1 or SEQ ID NO:3.In one aspect, the isolated nucleic acid molecule comprises a nucleicacid sequence which is SEQ ID NO:5 or SEQ ID NO:7.

One embodiment of the invention relates to an isolated nucleic acidmolecule comprising a nucleic acid sequence encoding a polypeptide thatis at least 50% (e.g., at least 55%; at least 60%; at least 65%; atleast 70%; at least 75%; at least 80%; at least 81%; at least 82%; atleast 83%; at least 84%; at least 85%; at least 86%; at least 87%; atleast 88%; at least 89%; at least 90%; at least 91%; at least 92%; atleast 93%; at least 94%; at least 95%; at least 96%; at least 97%; atleast 98%; at least 99%) identity to the amino acid sequence of SEQ IDNO:2 or SEQ ID NO:4. In one aspect, the polypeptide enhances theenzymatic activity of a PUFA synthase. In one aspect, the isolatednucleic acid molecule comprising a nucleic acid sequence encodes apolypeptide having an amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4.In one aspect, the isolated nucleic acid molecule comprises a nucleicacid sequence which is SEQ ID NO:6 or SEQ ID NO:8.

The invention includes the expression of one or more enhancing factorproteins as described and exemplified herein with a PUFA synthase systemas described herein and with an exogenous PPTase and/or acyl-CoAsynthetase (ACS) to increase PUFA production and/or accumulation in aheterologous host.

PUFA Synthase Systems (PUFA PKS Systems)

Accordingly, the present invention is directed to the enhancing factorproteins for use in connection with a PUFA synthase systems. As usedherein, a PUFA synthase system (which may also be referred to as a PUFAPKS system, PUFA synthase PKS-like system, PUFA synthase, or PUFAsynthase enzyme) generally has the following identifying features: (1)it produces PUFAs, and particularly, long chain PUFAs, as a naturalproduct of the system; and (2) it produces those PUFAs de novo usingmalonyl-CoA as the carbon source. In addition, the ACP domains presentin the PUFA synthase enzymes require activation by attachment of acofactor (4-phosphopantetheine). Attachment of this cofactor is carriedout by phosphopantetheinyl transferases (PPTase). If the endogenousPPTases of the host organism are incapable of activating the PUFAsynthase ACP domains, then it is necessary to provide a PPTase that iscapable of carrying out that function. The HetI enzyme of Nostoc sp. isan exemplary and suitable PPTase for activating PUFA synthase ACPdomains. Reference to a PUFA synthase system refers collectively to allof the genes and their encoded products that work together to producePUFAs in an organism.

More specifically, a PUFA synthase system as referenced herein producespolyunsaturated fatty acids (PUFAs) and particularly, long chain PUFAs(LCPUFAs), as products. For example, an organism that endogenously(naturally) contains a PUFA synthase system makes PUFAs using thissystem. According to the present invention, PUFAs are fatty acids with acarbon chain length of at least 16 carbons, and more preferably at least18 carbons, and more preferably at least 20 carbons, and more preferably22 or more carbons, with at least 3 or more double bonds, and preferably4 or more, and more preferably 5 or more, and even more preferably 6 ormore double bonds, wherein all double bonds are in the cisconfiguration. Reference to long chain polyunsaturated fatty acids(LCPUFAs) herein more particularly refers to fatty acids of 18 and morecarbon chain length, and preferably 20 and more carbon chain length,containing 3 or more double bonds. LCPUFAs of the omega-6 seriesinclude: gamma-linolenic acid (C18:3), di-homo-gamma-linolenic acid(C20:3, n-6), arachidonic acid (C20:4, n-6), adrenic acid (also calleddocosatetraenoic acid or DTA) (C22:4, n-6), and docosapentaenoic acid(C22:5, n-6). The LCPUFAs of the omega-3 series include: alpha-linolenicacid (C18:3), eicosatrienoic acid (C20:3, n-3), eicosatetraenoic acid(C20:4, n-3), eicosapentaenoic acid (C20:5, n-3), docosapentaenoic acid(C22:5, n-3), and docosahexaenoic acid (C22:6, n-3). The LCPUFAs alsoinclude fatty acids with greater than 22 carbons and 4 or more doublebonds including but not limited to C28:8, n-3.

The enhancing factor proteins according to the present invention can beused in connection with a PUFA synthase system from either a prokaryoticorganism or an eukaryotic organism. Description of the PUFA synthasesystems of various organisms can be found, for example, in U.S. Pat. No.6,140,486; U.S. Pat. No. 6,566,583; Metz et al., Science 293:290-293(2001); U.S. Pat. No. 7,247,461; U.S. Pat. No. 7,211,418; U.S. Pat. No.7,217,856, U.S. Patent Application Publication No. 2010-0266564, and PCTPublication No. WO 2006/135866.

The domain architecture of various PUFA synthase systems from marinebacteria and members of Thraustochytrium, and the structural andfunctional characteristics of genes and proteins comprising such PUFAsynthase systems, have been described in detail (see, e.g., U.S. Pat.No. 6,140,486; U.S. Pat. No. 6,566,583; Metz et al., Science 293:290-293(2001); U.S. Pat. No. 7,247,461; U.S. Pat. No. 7,211,418; U.S. Pat. No.7,217,856, U.S. Patent Application Publication No. 2010-0266564, and PCTPublication No. WO 2006/135866).

The enzymatic activity of a PUFA synthase can be measured in the form ofPUFA accumulation rate and PUFA composition profile. Two exemplarymethods for measuring the activity of PUFA synthase are described inExample 1 of the present application. In those assays, radio labeledmalonyl CoA is used as substrate and is converted into PUFAs by the PUFAsynthase system. The amount and compositions of PUFAs produced aremeasured.

Phosphopantetheinyl Transferase (PPTase)

As discussed under the general guidelines for the production of PUFAs ina heterologous host above, in order to produce PUFAs, a PUFA synthasesystem must work with an accessory protein that transfers a4′-phosphopantetheinyl moiety from coenzyme A to the acyl carrierprotein (ACP) domain(s). Therefore, a PUFA synthase system can beconsidered to include at least one 4′-phosphopantetheinyl transferase(PPTase) domain, or such a domain can be considered to be an accessorydomain or protein to the PUFA synthase system. Structural and functionalcharacteristics of PPTases have been described in detail, for example,in U.S. Pat. No. 7,247,461; U.S. Pat. No. 7,211,418; and U.S. Pat. No.7,217,856.

According to the present invention, a domain or protein having4′-phosphopantetheinyl transferase (PPTase) biological activity(function) is characterized as the enzyme that transfers a4′-phosphopantetheinyl moiety from Coenzyme A to the acyl carrierprotein (ACP). This transfer to an invariant serine reside of the ACPactivates the inactive apo-form to the holo-form. In both polyketide andfatty acid synthesis, the phosphopantetheine group forms thioesters withthe growing acyl chains. The PPTases are a family of enzymes that havebeen well characterized in fatty acid synthesis, polyketide synthesis,and non-ribosomal peptide synthesis. The sequences of many PPTases areknown, and crystal structures have been determined (e.g., Reuter et al.,1999, EMBO J. 18(23):6823-6831) as well as mutational analysis of aminoacid residues important for activity (Mofid et al., 2004, Biochemistry,43(14):4128-4136).

Accordingly, one embodiment of the invention relates to a geneticallymodified host cell or microorganism, wherein the host cell ormicroorganism has been genetically modified to express a core PUFAsynthase system as described herein, and also a PPTase as describedherein. Suitable PPTases are described above and are also described inthe art. The PPTase may be expressed on the same or a differentconstruct as one or more of the nucleic acid molecules encoding the corePUFA synthase protein or proteins. In one aspect, the PPTase is theNostoc HetI.

Acyl-CoA Synthetases

Acyl-CoA synthetase (ACS) proteins catalyze the conversion of free fattyacids (FFAs), including long chain PUFA, to acyl-CoA. Numerous examplesof polypeptides having ACS activity are known in the art and may be usedin embodiments herein. For example, Schizochytrium sp. ATCC 20888possesses one or more ACSs that are capable of converting the free fattyacid products of its PUFA synthase into acyl-CoA. See, e.g., U.S. Pat.No. 7,759,548.

The ACS protein can be derived from an organism that endogenouslyexpresses a PUFA synthase system. Such organisms include, but are notlimited to, a Thraustochytrid. In one aspect, the isolated ACS isderived from organisms of the genera Schizochytrium, Thraustochytrium,or Ulkenia. In another aspect, the isolated ACS is derived fromSchizochytrium ATCC 20888. In another aspect, any ACS that functions inconjunction with any PUFA synthase system to increase the productionand/or accumulation of PUFAs in a host cell or organism can be used inthe present invention.

Genetically Modified Cells and Organisms

To produce significantly high yields of one or more desiredpolyunsaturated fatty acids or other bioactive molecules, an organism,preferably a microorganism, can be genetically modified to alter theactivity and particularly, the end product(s), of the PUFA synthasesystem in the microorganism, or to introduce a PUFA synthase system intothe microorganism. The present invention relates to methods to improveor enhance the effectiveness of such genetic modification andparticularly, to improve or enhance the production and/or accumulationof the end product of a PUFA synthase system, preferably PUFA(s).

Therefore, one embodiment of the present invention relates to agenetically modified organism, wherein the organism expresses a PUFAsynthase system, and wherein the organism has been genetically modifiedto express the enhancing factor protein(s) as described herein for theimprovement of the production and/or accumulation of PUFAs (or otherbioactive products of the PUFA synthase system) by the host. If the PUFAsynthase system is heterologous to the host, then the organism is alsopreferably genetically modified to express a PPTase as a PUFA synthaseaccessory protein, which is described in detail above. In someembodiments, the organism has been genetically modified to express theone or more enhancing factor proteins described herein, and preferably acombination of TE2 and TE3 proteins or their homologues or enzymaticallyactive fragments.

In one embodiment, if the PUFA synthase system is endogenous to thehost, the organism can be genetically modified to express heterologousenhancing factor protein(s) as described above which improves theproduction and/or accumulation of PUFAs (or another bioactive product ofthe PUFA synthase system) in the host organism.

In another embodiment, the host organisms can be genetically modified toexpress a heterologous PUFA synthase system. The PUFA synthase systemexpressed by the organism can include any PUFA synthase system, forexample, PUFA PKS systems that are entirely derived from aSchizochytrium 20888 PUFA synthase system, as well as PUFA synthasesystems that are produced by “mixing and matching” nucleic acidsequences encoding proteins and/or domains from different PUFA synthasesystems (e.g., by mixing Schizochytrium 20888 PUFA synthase proteinsand/or domains with PUFA synthase proteins and/or domains from, e.g.,Schizochytrium 9695, or those derived from the genera Thraustochytrium,Ulkenia, Shewanella, Moritella, and/or Photobacterium, etc.) and/or fromdifferent non-PUFA synthase systems (e.g., type I modular, type Iiterative, type II or type III PKS systems), where the proteins and/ordomains from different organisms are combined to form a complete,functional PUFA synthase system. PUFA synthase systems, includingcombining PUFA synthase genes or proteins from different organisms, aredescribed in detail in U.S. Pat. No. 6,140,486; U.S. Pat. No. 6,566,583;Metz et al., Science 293:290-293 (2001); U.S. Pat. No. 7,247,461; U.S.Pat. No. 7,211,418; U.S. Pat. No. 7,217,856, U.S. Patent ApplicationPublication No. 2010-0266564; and PCT Publication No. WO 2006/135866;supra). PUFA synthase genes and proteins are also disclosed in: U.S.Pat. No. 7,939,305; and U.S. Pat. No. 7,208,590. Each of theabove-identified disclosures, and the genes and proteins describedtherein, is incorporated herein by reference.

Accordingly, encompassed by the present invention are methods togenetically modify organisms by: expressing one or more exogenousenhancing factor proteins described herein, especially a combination ofenhancing factor proteins TE2 and TE3, and/or by genetically modifyingat least one nucleic acid sequence in the organism that encodes at leastone functional domain or protein (or biologically active fragment orhomologue thereof) of a PUFA synthase system, including, but not limitedto, any PUFA synthase system specifically described herein. In oneembodiment, any of the exogenously introduced nucleic acid sequences canbe optimized for codon usage or improved expression in the host. In oneembodiment, any of the introduced nucleic acid sequences can be targetedto one or more organelles in the organism. Various embodiments of suchsequences, methods to genetically modify an organism, specificmodifications, and combinations thereof have been described in detailabove and are encompassed here. Typically, the method is used to producea particular genetically modified organism that produces a particularbioactive molecule or molecules.

Preferred genetically modified organisms include genetically modifiedmicroorganisms.

Preferably, a genetically modified organism of the invention producesone or more polyunsaturated fatty acids including, but not limited to,DHA (C22:6, n-3), DPA (C22:5, n-6 or n-3), EPA (C20:5, n-3), ARA (C20:4,n-6), GLA (C18:3, n-6), ALA (C18:3, n-3), and/or SDA (C18:4, n-3)), andmore preferably, one or more longer chain PUFAs, including, but notlimited to, DHA (C22:6, n-3), DPA (C22:5, n-6 or n-3), EPA (C20:5, n-3),or DTA (C22:4, n-6), or any combination thereof. In a particularlypreferred embodiment, a genetically modified microorganism of theinvention produces one or more polyunsaturated fatty acids including,but not limited to, DHA (C22:6, n-3) and DPA (C22:5, n-6 or n-3), or anycombination thereof.

According to the present invention, a genetically modified organismincludes an organism that has been modified using recombinant technologyor by classical mutagenesis and screening techniques. As used herein,genetic modifications that result in a decrease in gene expression, inthe function of the gene, or in the function of the gene product (i.e.,the protein encoded by the gene) can be referred to as inactivation(complete or partial), deletion, interruption, blockage ordown-regulation of a gene. For example, a genetic modification in a genewhich results in a decrease in the function of the protein encoded bysuch gene, can be the result of a complete deletion of the gene (i.e.,the gene does not exist, and therefore the protein does not exist), amutation in the gene which results in incomplete or no translation ofthe protein (e.g., the protein is not expressed), or a mutation in thegene which decreases or abolishes the natural function of the protein(e.g., a protein is expressed which has decreased or no enzymaticactivity or action). Genetic modifications that result in an increase ingene expression or function can be referred to as amplification,overproduction, overexpression, activation, enhancement, addition, orup-regulation of a gene.

Genetically Modified Microorganisms

As used herein, a genetically modified microorganism can include agenetically modified bacterium, protist, microalgae, algae, fungus, orother microbe. Such a genetically modified microorganism has a genomewhich is modified (i.e., mutated or changed) from its normal (i.e.,wild-type or naturally occurring) form such that the desired result isachieved (i.e., increased or modified PUFA synthase activity and/orproduction and accumulation of a desired product using the PUFA PKSsystem). Genetic modification of a microorganism can be accomplishedusing classical strain development and/or molecular genetic techniques.Such techniques known in the art and are generally disclosed formicroorganisms, for example, in Sambrook et al., 1989, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Labs Press. Thereference Sambrook et al., ibid., is incorporated by reference herein inits entirety. A genetically modified microorganism can include amicroorganism in which nucleic acid molecules have been inserted,deleted or modified (i.e., mutated; e.g., by insertion, deletion,substitution, and/or inversion of nucleotides), in such a manner thatsuch modifications provide the desired effect within the microorganism.

Examples of suitable host microorganisms for genetic modificationinclude, but are not limited to, yeast including Saccharomycescerevisiae, Saccharomyces carlsbergensis, or other yeast such asCandida, Kluyveromyces, or other fungi, for example, filamentous fungisuch as Aspergillus, Neurospora, Penicillium, etc. Bacterial cells alsomay be used as hosts. These include, but are not limited to, Escherichiacoli, which can be useful in fermentation processes. Alternatively, andonly by way of example, a host such as a Lactobacillus species orBacillus species can be used as a host.

Other hosts for use in the present invention include microorganisms froma genus including, but not limited to: Thraustochytrium, Japonochytrium,Aplanochytrium, Elina and Schizochytrium within the Thraustochytriaceae,and Labyrinthula, Labyrinthuloides, and Labyrinthomyxa within theLabyrinthulaceae. Preferred species within these genera include, but arenot limited to: any species described below. Particularly preferredstrains of Thraustochytriales include, but are not limited to:Schizochytrium sp. (S31) (ATCC 20888); Schizochytrium sp. (ATCCPTA-9695): Schizochytrium sp. (S8) (ATCC 20889); Schizochytrium sp.(LC-RM) (ATCC 18915); Schizochytrium sp. (SR21); Schizochytrium sp.N230D, Schizochytrium aggregatum (Goldstein et Belsky) (ATCC 28209);Schizochytrium limacinum (Honda et Yokochi) (IFO 32693);Thraustochytrium sp. (23B) (ATCC 20891 or ATCC 20892); Thraustochytriumstriatum (Schneider) (ATCC 24473); Thraustochytrium aureum (Goldstein)(ATCC 34304); Thraustochytrium roseum (Goldstein) (ATCC 28210); andJaponochytrium sp. (L1) (ATCC 28207).

In one embodiment of the present invention, the enhancing factorproteins of PUFA synthase (e.g., TE2 and TE3) of a microorganism areexogenously introduced into a host microorganism which has an endogenousPUFA synthase system to increase the amount of PUFAs produced. Inanother embodiment, the exogenous enhancing factor proteins and anexogenous PUFA synthase system of a microorganism are introduced into ahost microorganism which does not have any PUFA synthase system toproduce detectable amount of PUFAs. Examples of heterologous sequencesthat could be introduced into a host genome include sequences encodingat least one functional PUFA synthase domain or protein from another PKSsynthase or even an entire PUFA synthase system (e.g., all genesassociated with the PUFA synthase system). A heterologous sequence canalso include a sequence encoding a modified functional domain (ahomologue) of a natural domain from a PUFA synthase system. Otherheterologous sequences that can be introduced into the host genomeinclude PPTase and/or ACS.

Therefore, it is an object of the present invention to produce, via thegenetic manipulation of microorganisms as described herein, PUFAs and,by extension, oils obtained from such microorganisms comprising thesePUFAs. Examples of PUFAs that can be produced by the present inventioninclude, but are not limited to, DHA (docosahexaenoic acid (C22:6,n-3)), DPA (docosapentaenoic acid (C22:5, n-6 or n-3)), and EPA(eicosapentaenoic acid (C20:5, n-3)) and any combinations thereof. Thepresent invention allows for the production of commercially valuablelipids enriched in one or more desired (primary) PUFAs by the presentinventors' development of genetically modified microorganisms throughthe use of the PUFA synthase system that produces PUFAs.

When using a PUFA synthase system as preferred in the present invention,a given PUFA synthase system derived from a particular organism willproduce particular PUFA(s), such that selection of a PUFA synthasesystem from a particular organism will result in the production ofspecified PUFAs. For example, use of a PUFA synthase system fromSchizochytrium 20888 will result in the production of DHA and DPAn-6 asthe primary PUFAs.

Schizochytrium 20888 can accumulate high levels of oil (>60% of thebiomass) and DHA can comprise >40% of the fatty acids present in thatbiomass. In the native organism the DHA to DPAn-6 ratio typically rangesbetween 2.3 to 2.7. Expression of the PUFA synthase subunits ofSchizochytrium 20888, along with an appropriate PPTase (e.g., HetI fromNostoc sp.) in heterologous host cells has resulted in production of DHAand DPAn-6 in those cells. Although DHA and DPAn-6 are produced in cellsof E. coli, and in yeast and higher plants, the levels have notapproached those observed in the native organism. Additionally, in bothyeast and in plants, the DHA to DPAn-6 ratio is typically significantlylower than that observed in the native organism.

According to the present invention, a microorganism can be geneticallymodified to introduce one or more genes encoding the enhancing factorproteins described herein to increase the production of PUFAs from theparticular PUFA synthase present in that organism (e.g., DHA, DPAn-6and/or EPA). In addition, introduction of the enhancing factor proteinsdescribed herein can result in an alteration of the relative amounts ofthe PUFAs produced by the particular PUFA synthase present in thatorganism (e.g., the ratio of DHA to DPAn-6 produced in the microorganismmay be increased).

Therefore, one embodiment of the present invention relates to agenetically modified microorganism (e.g., wherein the microorganism hasbeen genetically modified to express a PUFA synthase system describedherein), which includes the core PUFA synthase, a PPTase, and/or an ACS,as described herein, wherein the microorganism has been furthergenetically modified to express one or more enhancing factor proteins asdescribed herein for the improvement of the production and/oraccumulation of PUFAs (or other bioactive products of the PUFA synthasesystem) by the host. Preferably, such enhancing factor protein is acombination of TE2 and TE3 proteins. The enhancing factor proteinsdescribed here, such as TE2 and TE3, include homologues and biologicallyactive fragments of such proteins.

In some embodiments, the genetically modified microorganism which isgenetically modified to express a heterologous PUFA synthase system anda combination of TE2 and TE3 proteins has an increased level of totalPUFA accumulation relative to the microorganism without the expressionof TE2 and TE3 proteins. In one embodiment, the total PUFA accumulationis increased more than 2-fold, more than 3-fold, more than 4-fold, morethan 5-fold, more than 6-fold, more than 7-fold, more than 8-fold, morethan 9-fold, or more than 10-fold. In one embodiment, the increasedlevel of total PUFA accumulation is close or equal to the level producedby the native microorganism of the heterologous PUFA synthase system. Inone embodiment, the increased level of total PUFA accumulation isbetween 90% and 110% of the amount of PUFA produced by the nativemicroorganism of the heterologous PUFA synthase system.

In some embodiments, the genetically modified microorganism which isgenetically modified to express a heterologous PUFA synthase system anda combination of TE2 and TE3 proteins has an increased level of totalDHA, DPA(n-6), and EPA accumulation when compared to the microorganismwithout the expression of TE2 and TE3 proteins. In one embodiment, thelevel of DHA and DPAn-6 accumulation is increased more than 2-fold, morethan 3-fold, more than 4-fold, more than 5-fold, more than 6-fold, morethan 7-fold, more than 8-fold, more than 9-fold, or more than 10-fold.In one embodiment, the increased level of DHA and DPAn-6 accumulation isclose or equal to the level produced by the native microorganism of theheterologous PUFA synthase system. In one embodiment, the increasedlevel of DHA and DPAn-6 accumulation is between 90% and 110% of theamount of PUFA produced by the native microorganism of the heterologousPUFA synthase system.

In some embodiments, the genetically modified microorganism which isgenetically modified to express a heterologous PUFA synthase system anda combination of TE2 and TE3 proteins has an enhanced ratio ofDHA:DPAn-6 by weight of total fatty acids than the microorganism withoutsuch genetic modification. In one embodiment, the higher ratio ofDHA:DPAn-6 is close or equal to the ratio in the microorganism where thePUFA synthase system is derived from. In one embodiment, the enhancedratio of DHA:DPAn-6 is between 90% and 110% of the ratio of DHA:DPAn-6of the microorganism where the PUFA synthase system is derived from.

The genetic modification of a microorganism according to the presentinvention can be made either to positively affect the activity of thePUFA synthase system expressed by the microorganism, or to negativelyaffect the activity of the PUFA synthase system expressed by themicroorganism. For example, the activity of the PUFA synthase system canbe reduced or even blocked by a reduction or elimination of theexpression of the endogenous enhancing factor proteins of the hostmicroorganism. In some embodiments, the host microorganism is aThraustochytriales microorganism. In one embodiment, the hostmicroorganism is a Schizochytrium 20888. In another embodiment, the hostmicroorganism is a Schizochytrium 9695.

Uses for Genetically Modified Organisms of the Invention

One embodiment of the present invention is a method to produce desiredbioactive molecules (also referred to as products or compounds) bygrowing a genetically modified microorganism of the present invention(described in detail above). Preferably, the bioactive molecule is aPUFA, and most preferably, an LCPUFA. Preferably, the geneticallymodified microorganism is a genetically modified microorganism. Such amethod includes, for example, the step of culturing in a fermentationmedium a microorganism as described previously herein and in accordancewith the present invention. Preferred host cells and microorganisms forgenetic modification related to the PUFA synthase system of theinvention are described above.

One embodiment of the present invention is a method to produce desiredPUFAs by culturing a genetically modified microorganism of the presentinvention (described in detail above). Such a method includes the stepof culturing in a fermentation medium and under conditions effective toproduce the PUFA(s) a microorganism that has a genetic modification asdescribed previously herein and in accordance with the presentinvention. An appropriate, or effective, medium refers to any medium inwhich a genetically modified microorganism of the present invention,when cultured, is capable of producing the desired PUFA product(s). Sucha medium is typically an aqueous medium comprising assailable carbon,nitrogen and phosphate sources. Such a medium can also includeappropriate salts, minerals, metals and other nutrients. Anymicroorganisms of the present invention can be cultured in conventionalfermentation bioreactors. The microorganisms can be cultured by anyfermentation process which includes, but is not limited to, batch,fed-batch, cell recycle, and continuous fermentation. Preferred growthconditions for Thraustochytrid microorganisms according to the presentinvention are well known in the art and are described in detail, forexample, in U.S. Pat. No. 5,130,242, U.S. Pat. No. 5,340,742, and U.S.Pat. No. 5,698,244, each of which is incorporated herein by reference inits entirety.

The desired PUFA(s) and/or other bioactive molecules produced by thegenetically modified microorganism can be recovered from thefermentation medium using conventional separation and purificationtechniques. For example, the fermentation medium can be filtered orcentrifuged to remove microorganisms, cell debris and other particulatematter, and the product can be recovered from the cell-free supernatantby conventional methods, such as, for example, ion exchange,chromatography, extraction, solvent extraction, phase separation,membrane separation, electrodialysis, reverse osmosis, distillation,chemical derivatization and crystallization. Alternatively,microorganisms producing the PUFA(s), or extracts and various fractionsthereof, can be used without removal of the microorganism componentsfrom the product.

The invention further includes any microorganisms described herein aswell as any oils produced by the microorganisms described herein. Theinvention also includes any products produced using the microorganismsor oils described herein.

One embodiment of the present invention relates to a method to modify aproduct containing at least one fatty acid, comprising adding to theproduct a microorganism or oil produced by a genetically modifiedmicroorganism according to the invention and as described herein (e.g.,a microorganism that has been genetically modified with a PUFA synthasesystem, makes use of any of the strategies for improvement of productionand/or accumulation of PUFAs described herein, and has a fatty acidprofile described herein). Any products produced by this method orgenerally containing any microorganisms or oils from the microorganismsdescribed herein are also encompassed by the invention.

Preferably, the product is selected from the group consisting of a food,a dietary supplement, a pharmaceutical formulation, a feedstuff, ahumanized animal milk, and an infant formula. Suitable pharmaceuticalformulations include, but are not limited to, an anti-inflammatoryformulation, a chemotherapeutic agent, an active excipient, anosteoporosis drug, an anti-depressant, an anti-convulsant, ananti-Heliobactor pylori drug, a drug for treatment of neurodegenerativedisease, a drug for treatment of degenerative liver disease, anantibiotic, and a cholesterol lowering formulation. In one embodiment,the product is used to treat a condition selected from the groupconsisting of: chronic inflammation, acute inflammation,gastrointestinal disorder, cancer, cachexia, cardiac restenosis,neurodegenerative disorder, degenerative disorder of the liver, bloodlipid disorder, osteoporosis, osteoarthritis, autoimmune disease,preeclampsia, preterm birth, age related maculopathy, pulmonarydisorder, and peroxisomal disorder.

In some embodiments of the invention, the PUFAs produced by thegenetically modified organisms or the methods disclosed in the presentinvention can be incorporated into a component of food or feed (e.g., afood supplement). Types of food products into which the PUFAs can beincorporated according to the present invention are not particularlylimited, and include food products such as fine bakery wares, bread androlls, breakfast cereals, processed and unprocessed cheese, condiments(ketchup, mayonnaise, etc.), dairy products (milk, yogurt), puddings andgelatine desserts, carbonated drinks, teas, powdered beverage mixes,processed fish products, fruit-based drinks, chewing gum, hardconfectionery, frozen dairy products, processed meat products, nut andnut-based spreads, pasta, processed poultry products, gravies andsauces, potato chips and other chips or crisps, chocolate and otherconfectionery, soups and soup mixes, soya based products (milks, drinks,creams, whiteners), vegetable oil-based spreads, and vegetable-baseddrinks.

Examples of feedstuffs into which the PUFAs produced in accordance withthe present invention may be incorporated include, for instance, petfoods such as cat foods, dog foods and the like, feeds for aquariumfish, cultured fish or crustaceans, etc., feed for farm-raised animals(including livestock and further including fish or crustaceans raised inaquaculture). The PUFA containing genetically modified organismsproduced in accordance with the present invention, such as thegenetically modified microorganisms, may be incorporated directly intofeed products.

GENERAL DEFINITIONS AND GUIDANCE

According to the present invention, an “isolated” protein or nucleicacid is a protein or nucleic acid that has been substantially separated,produced apart from or purified away from other biological components inthe cell of the organism in which the protein or the nucleic acidsnaturally occurs. “Isolated” effects a chemical or functional change inthe protein or nucleic acid (e.g., a nucleic acid may be isolated from achromosome by breaking chemical bonds connecting the nucleic acid to theremaining DNA in the chromosome). Nucleic acid molecules and proteinsthat have been “isolated” include nucleic acid molecules and proteinspurified by standard purification methods. “Isolated” does not reflectthe extent to which the protein or nucleic acid has been purified. Theterm also embraces nucleic acids and proteins prepared by recombinantexpression in a host cell, as well as chemically-synthesized nucleicacid molecules, proteins, and peptides. As used herein, the term“nucleic acid molecule” may refer to a polymeric form of nucleotides,which may include both sense and anti-sense strands of RNA, cDNA,genomic DNA, and synthetic forms and mixed polymers of the above. Anucleotide may refer to a ribonucleotide, deoxyribonucleotide, or amodified form of either type of nucleotide. A “nucleic acid molecule” asused herein is synonymous with “nucleic acid” and “polynucleotide.” Anucleic acid molecule is usually at least 10 bases in length, unlessotherwise specified. The term includes single- and double-stranded formsof DNA. A nucleic acid molecule can include either or both naturallyoccurring and modified nucleotides linked together by naturallyoccurring and/or non-naturally occurring nucleotide linkages.

According to the present invention, a “recombinant” nucleic acid is anucleic acid that is constructed by joining two or more nucleic acidmolecules and that can replicate in a living cell. One embodiment of thepresent invention includes a recombinant nucleic acid moleculecomprising a recombinant vector and a nucleic acid sequence describedabove. Such nucleic acid sequence encodes a protein or peptide having abiological activity of any of the EF-X proteins described above.According to the present invention, a recombinant vector is anengineered (i.e., artificially produced) nucleic acid molecule that isused as a tool for manipulating a nucleic acid sequence of choice andfor introducing such a nucleic acid sequence into a host cell. Therecombinant vector is therefore suitable for use in cloning, sequencing,and/or otherwise manipulating the nucleic acid sequence of choice, suchas by expressing and/or delivering the nucleic acid sequence of choiceinto a host cell to form a recombinant cell. Such a vector typicallycontains heterologous nucleic acid sequences, that is nucleic acidsequences that are not naturally found adjacent to nucleic acid sequenceto be cloned or delivered, although the vector can also containregulatory nucleic acid sequences (e.g., promoters, untranslatedregions) which are naturally found adjacent to nucleic acid molecules ofthe present invention or which are useful for expression of the nucleicacid molecules of the present invention (discussed in detail below). Thevector can be either RNA or DNA, either prokaryotic or eukaryotic, andtypically is a plasmid. The vector can be maintained as anextrachromosomal element (e.g., a plasmid) or it can be integrated intothe chromosome of a recombinant organism (e.g., a microbe or a plant).The entire vector can remain in place within a host cell, or undercertain conditions, the plasmid DNA can be deleted, leaving behind thenucleic acid molecule of the present invention. The integrated nucleicacid molecule can be under chromosomal promoter control, under native orplasmid promoter control, or under a combination of several promotercontrols. Single or multiple copies of the nucleic acid molecule can beintegrated into the chromosome. A recombinant vector of the presentinvention can contain at least one selectable marker.

In one embodiment, a recombinant vector used in a recombinant nucleicacid molecule of the present invention is an expression vector. As usedherein, the phrase “expression vector” is used to refer to a vector thatis suitable for production of an encoded product (e.g., a protein ofinterest). In this embodiment, a nucleic acid sequence encoding theproduct to be produced (e.g., a PUFA PKS domain or protein) is insertedinto the recombinant vector to produce a recombinant nucleic acidmolecule. The nucleic acid sequence encoding the protein to be producedis inserted into the vector in a manner that operatively links thenucleic acid sequence to regulatory sequences in the vector that enablethe transcription and translation of the nucleic acid sequence withinthe recombinant host cell.

In another embodiment, a recombinant vector used in a recombinantnucleic acid molecule of the present invention is a targeting vector. Asused herein, the phrase “targeting vector” is used to refer to a vectorthat is used to deliver a particular nucleic acid molecule into arecombinant host cell, wherein the nucleic acid molecule is used todelete, inactivate, or replace an endogenous gene or portion of a genewithin the host cell or microorganism (i.e., used for targeted genedisruption or knock-out technology). Such a vector may also be known inthe art as a “knock-out” vector. In one aspect of this embodiment, aportion of the vector, but more typically, the nucleic acid moleculeinserted into the vector (i.e., the insert), has a nucleic acid sequencethat is homologous to a nucleic acid sequence of a target gene in thehost cell (i.e., a gene which is targeted to be deleted or inactivated).The nucleic acid sequence of the vector insert is designed to associatewith the target gene such that the target gene and the insert mayundergo homologous recombination, whereby the endogenous target gene isdeleted, inactivated, attenuated (i.e., by at least a portion of theendogenous target gene being mutated or deleted), or replaced. The useof this type of recombinant vector to replace an endogenousSchizochytrium gene, for example, with a recombinant gene has beenpreviously described by the present inventors, and the general techniquefor genetic transformation of Thraustochytrids is described in detail inU.S. Pat. No. 7,001,772.

As used herein, the term “lipid” includes phospholipids; free fattyacids; esters of fatty acids; triacylglycerols; diacylglycerides;monoacylglycerides; lysophospholipids; soaps; phosphatides; waxes(esters of alcohols and fatty acids); sterols and sterol esters;carotenoids; xanthophylls (e.g., oxycarotenoids); hydrocarbons; andother lipids known to one of ordinary skill in the art.

Reference to a particular protein from a specific organism or to aparticular protein being derived from a specific organism, such as a“TE2 derived from Schizochytrium sp. ATCC-20888” or “Sz-TE2”, by way ofexample, refers to a TE2 (including a homologue of the naturallyoccurring TE2) from a Schizochytrium sp. ATCC-20888 or a Sz-TE2 that hasbeen otherwise produced from the knowledge of the structure (e.g.,sequence) of a naturally occurring TE2 from Schizochytrium. In otherwords, a Sz-TE2 includes any TE2 that has the structure and function ofa naturally occurring TE2 from Schizochytrium or that has a structureand function that is sufficiently similar to a TE2 from Schizochytriumsuch that the TE2 is a biologically active (i.e., has biologicalactivity) homologue of a naturally occurring TE2 from Schizochytrium sp.As such, a Sz-TE2 can include purified, partially purified, recombinant,mutated/modified and synthetic proteins.

According to the present invention, the terms “modification” and“mutation” can be used interchangeably, particularly with regard to themodifications/mutations to the primary amino acid sequences of a proteinor peptide (or nucleic acid sequences) described herein. The term“modification” can also be used to describe post-translationalmodifications to a protein or peptide including, but not limited to,methylation, farnesylation, carboxymethylation, geranyl geranylation,glycosylation, phosphorylation, acetylation, myristoylation,prenylation, palmitation, and/or amidation. Modifications can alsoinclude, for example, complexing a protein or peptide with anothercompound. Such modifications can be considered to be mutations, forexample, if the modification is different than the post-translationalmodification that occurs in the natural, wild-type protein or peptide.

As used herein, the term “homologue” is used to refer to a protein orpeptide which differs from a naturally occurring protein or peptide(i.e., the “prototype” or “wild-type” protein) by one or more minormodifications or mutations to the naturally occurring protein orpeptide, but which maintains the overall basic protein and side chainstructure of the naturally occurring form (i.e., such that the homologueis identifiable as being related to the wild-type protein). Such changesinclude, but are not limited to: changes in one or a few (e.g., 1% orless) amino acid side chains; changes in one or a few (e.g., 1% or less)amino acids, including deletions (e.g., a truncated version of theprotein or peptide) insertions and/or substitutions; changes instereochemistry of one or a few (e.g., 1% or less) atoms; and/or minorderivatizations, including but not limited to: methylation,farnesylation, geranyl geranylation, glycosylation, carboxymethylation,phosphorylation, acetylation, myristoylation, prenylation, palmitation,and/or amidation. A homologue can have either enhanced, decreased, orsubstantially similar properties as compared to the naturally occurringprotein or peptide. Preferred homologues of a protein are described indetail below. It is noted that homologues can include syntheticallyproduced homologues, naturally occurring allelic variants of a givenprotein or domain thereof, or homologous sequences from organisms otherthan the organism from which the reference sequence was derived.

Conservative substitutions typically include substitutions within thefollowing groups: glycine and alanine; valine, isoleucine and leucine;aspartic acid, glutamic acid, asparagine, and glutamine; serine andthreonine; lysine and arginine; and phenylalanine and tyrosine.Substitutions may also be made on the basis of conserved hydrophobicityor hydrophilicity (Kyte and Doolittle, J. Mol. Biol. 157:105 (1982)), oron the basis of the ability to assume similar polypeptide secondarystructure (Chou and Fasman, Adv. Enzymol. 47: 45 (1978)).

Homologues can be the result of natural allelic variation or naturalmutation. A naturally occurring allelic variant of a nucleic acidencoding a protein is a gene that occurs at essentially the same locus(or loci) in the genome as the gene which encodes such protein, butwhich, due to natural variations caused by, for example, mutation orrecombination, has a similar but not identical sequence. Allelicvariants typically encode proteins having similar activity to that ofthe protein encoded by the gene to which they are being compared. Oneclass of allelic variants can encode the same protein but have differentnucleic acid sequences due to the degeneracy of the genetic code.Allelic variants can also comprise alterations in the 5′ or 3′untranslated regions of the gene (e.g., in regulatory control regions).Allelic variants are well known to those skilled in the art.

Homologues can be produced using techniques known in the art for theproduction of proteins including, but not limited to, directmodifications to the isolated, naturally occurring protein, directprotein synthesis, or modifications to the nucleic acid sequenceencoding the protein using, for example, classic or recombinant DNAtechniques to effect random or targeted mutagenesis.

Modifications or mutations in protein homologues, as compared to thewild-type protein, either increase, decrease, or do not substantiallychange, the basic biological activity of the homologue as compared tothe naturally occurring (wild-type) protein. In general, the biologicalactivity or biological action of a protein refers to any function(s)exhibited or performed by the protein that is ascribed to the naturallyoccurring form of the protein as measured or observed in vivo (i.e., inthe natural physiological environment of the protein) or in vitro (i.e.,under laboratory conditions). Biological activities of PUFA PKS systemsand the individual proteins/domains that make up a PUFA synthase systemhave been described in detail elsewhere herein and in the referencedpatents and applications.

Modifications of a protein, such as in a homologue, may result inproteins having the same biological activity as the naturally occurringprotein, or in proteins having decreased or increased biologicalactivity as compared to the naturally occurring protein. Modificationswhich result in a decrease in protein expression or a decrease in theactivity of the protein, can be referred to as inactivation (complete orpartial), down-regulation, or decreased action (or activity) of aprotein. Similarly, modifications which result in an increase in proteinexpression or an increase in the activity of the protein, can bereferred to as amplification, overproduction, activation, enhancement,up-regulation or increased action (or activity) of a protein. It isnoted that general reference to a homologue having the biologicalactivity of the wild-type protein does not necessarily mean that thehomologue has identical biological activity as the wild-type protein,particularly with regard to the level of biological activity. Rather, ahomologue can perform the same biological activity as the wild-typeprotein, but at a reduced or increased level of activity as compared tothe wild-type protein. A functional domain of a protein is a domain(i.e., a domain can be a portion of a protein) that is capable ofperforming a biological function (i.e., has biological activity).

As used herein, the term “biologically active fragment” refers to aportion of a protein which such fragment maintains at least part of thebiological activity of the protein. For example, the biological activefragment of TE2 is a portion of TE2 and it maintains at least part ofthe biological activity of TE2 which enhances the enzymatic activity ofPUFA synthase in the host cell where TE2 is exogenously expressed.

The term “PUFA synthase” as used herein refers to an enzyme thatproduces PUFAs (e.g., LCPUFAs), as well as a domain of such an enzyme.Some specific PUFA synthases are designated herein by an additionalnotation (e.g., “Schizochytrium 20888 PUFA synthase”). The term “PUFAsynthase system” refers to one or more PUFA synthase(s) and anyaccessory enzyme that can affect the function of the PUFA synthase(e.g., a PPTase). For example, the PUFA synthase system inSchizochytrium 20888 consists of the native PUFA synthase subunits andthe native PPTase.

As used herein, the term “transformation” or “transduction” refers tothe transfer of one or more nucleic acid molecule(s) into a cell. A cellis “transformed” by a nucleic acid molecule transduced into the cellwhen the nucleic acid molecule becomes stably replicated by the cell,either by incorporation of the nucleic acid molecule into the cellulargenome, or by episomal replication. As used herein, the term“transformation” encompasses all techniques by which a nucleic acidmolecule can be introduced into such a cell.

The term “sequence identity” or “identity,” as used herein in thecontext of two nucleic acid or polypeptide sequences, may refer to theresidues in the two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. As used herein, theterm “percentage of sequence identity” may refer to the value determinedby comparing two optimally aligned sequences (e.g., nucleic acidsequences, and amino acid sequences) over a comparison window, whereinthe portion of the sequence in the comparison window may compriseadditions or deletions (i.e., gaps) as compared to the referencesequence (which does not comprise additions or deletions) for optimalalignment of the two sequences. The percentage is calculated bydetermining the number of positions at which the identical nucleotide oramino acid residue occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the comparison window, and multiplying the resultby 100 to yield the percentage of sequence identity.

Methods for aligning sequences for comparison are well-known in the art.Various programs and alignment algorithms are described in, for example:Smith and Waterman (1981) Adv. Appl. Math. 2:482; Needleman and Wunsch(1970) J. Mol. Biol. 48:443; Pearson and Lipman (1988) Proc. Natl. Acad.Sci. U.S.A. 85:2444; Higgins and Sharp (1988) Gene 73:237-44; Higginsand Sharp (1989) CABIOS 5:151-3; Corpet et al. (1988) Nucleic Acids Res.16:10881-90; Huang et al. (1992) Comp. Appl. Biosci. 8:155-65; Pearsonet al. (1994) Methods Mol. Biol. 24:307-31; Tatiana et al. (1999) FEMSMicrobiol. Lett. 174:247-50. A detailed consideration of sequencealignment methods and homology calculations can be found in, e.g.,Altschul et al. (1990) J. Mol. Biol. 215:403-10.

The National Center for Biotechnology Information (NCBI) Basic LocalAlignment Search Tool (BLAST™; Altschul et al. (1990)) is available fromseveral sources, including the National Center for BiotechnologyInformation (Bethesda, Md.), and on the internet, for use in connectionwith several sequence analysis programs. A description of how todetermine sequence identity using this program is available on theinternet under the “help” section for BLAST™. For comparisons of nucleicacid sequences, the “Align two or more sequences” function of the BLAST™(blastn) program may be employed using the default parameters. Forcomparisons of protein sequences, the “Align two or more sequences”function of the BLAST™ (blastp) program may be employed using thedefault parameters. As used herein, hybridization conditions refer tostandard hybridization conditions under which nucleic acid molecules areused to identify similar nucleic acid molecules. Such standardconditions are disclosed, for example, in Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Labs Press (1989).Sambrook et al., ibid., is incorporated by reference herein in itsentirety (see specifically, pages 9.31-9.62). In addition, formulae tocalculate the appropriate hybridization and wash conditions to achievehybridization permitting varying degrees of mismatch of nucleotides aredisclosed, for example, in Meinkoth et al., Anal. Biochem. 138, 267(1984); Meinkoth et al., ibid., incorporated by reference herein in itsentirety.

More particularly, moderate stringency hybridization and washingconditions, as referred to herein, refer to conditions which permitisolation of nucleic acid molecules having at least about 70% nucleicacid sequence identity with the nucleic acid molecule being used toprobe in the hybridization reaction (i.e., conditions permitting about30% or less mismatch of nucleotides). High stringency hybridization andwashing conditions, as referred to herein, refer to conditions whichpermit isolation of nucleic acid molecules having at least about 80%nucleic acid sequence identity with the nucleic acid molecule being usedto probe in the hybridization reaction (i.e., conditions permittingabout 20% or less mismatch of nucleotides). Very high stringencyhybridization and washing conditions, as referred to herein, refer toconditions which permit isolation of nucleic acid molecules having atleast about 90% nucleic acid sequence identity with the nucleic acidmolecule being used to probe in the hybridization reaction (i.e.,conditions permitting about 10% or less mismatch of nucleotides). Asdiscussed above, one of skill in the art can use the formulae inMeinkoth et al., ibid. to calculate the appropriate hybridization andwash conditions to achieve these particular levels of nucleotidemismatch. Such conditions will vary, depending on whether DNA:RNA orDNA:DNA hybrids are being formed. Calculated melting temperatures forDNA:DNA hybrids are 10° C. less than for DNA:RNA hybrids. In particularembodiments, stringent hybridization conditions for DNA:DNA hybridsinclude hybridization at an ionic strength of 6×SSC (0.9 M Na⁺) at atemperature of between about 20° C. and about 35° C. (lower stringency),more preferably, between about 28° C. and about 40° C. (more stringent),and even more preferably, between about 35° C. and about 45° C. (evenmore stringent), with appropriate wash conditions. In particularembodiments, stringent hybridization conditions for DNA:RNA hybridsinclude hybridization at an ionic strength of 6×SSC (0.9 M Na⁺) at atemperature of between about 30° C. and about 45° C., more preferably,between about 38° C. and about 50° C., and even more preferably, betweenabout 45° C. and about 55° C., with similarly stringent wash conditions.These values are based on calculations of a melting temperature formolecules larger than about 100 nucleotides, 0% formamide and a G+Ccontent of about 40%. Alternatively, T_(m) can be calculated empiricallyas set forth in Sambrook et al., supra, pages 9.31 to 9.62. In general,the wash conditions should be as stringent as possible, and should beappropriate for the chosen hybridization conditions. For example,hybridization conditions can include a combination of salt andtemperature conditions that are approximately 20-25° C. below thecalculated T_(m) of a particular hybrid, and wash conditions typicallyinclude a combination of salt and temperature conditions that areapproximately 12-20° C. below the calculated T_(m) of the particularhybrid. One example of hybridization conditions suitable for use withDNA:DNA hybrids includes a 2-24 hour hybridization in 6×SSC (50%formamide) at about 42° C., followed by washing steps that include oneor more washes at room temperature in about 2×SSC, followed byadditional washes at higher temperatures and lower ionic strength (e.g.,at least one wash as about 37° C. in about 0.1×-0.5×SSC, followed by atleast one wash at about 68° C. in about 0.1×-0.5×SSC).

Each publication, patent or patent application referenced herein isincorporated herein by reference in its entirety.

The following examples are provided for the purpose of illustration andare not intended to limit the scope of the present invention.

EXAMPLES Example 1

The following example describes the development of an assay used toidentify factors which may enhance the enzymatic activity of PUFAsynthases.

This in vitro assay was developed to identify any factors present inhomogenates of cells of Schizochytrium 20888, which could facilitate theactivity of a PUFA synthase system. The assay was based on anobservation that addition of an extract derived from a strain ofSchizochytrium 20888 to a homogenate of E. coli expressing theSchizochytrium 20888 PUFA synthase (plus HetI) resulted in an increasein the in vitro activity of that PUFA synthase system (see the left twosamples of FIG. 2, i.e., BUFF K and Q-KO). The general features of thisassay are described below.

Preparation of homogenates of an E. coli strain expressing theSchizochytrium 20888 PUFA synthase for use in the PUFA synthaseenhancement assay: the Schizochytrium 20888 PUFA synthase subunits andHetI (from Nostoc sp.) were expressed in E. coli strain JK824 asdescribed in Hauvermale et al., 2008, Lipids, 41:739-747 and Metz etal., US Patent Application Publication No. 2013-0150599. The PUFAsynthase subunit genes were expressed as a synthetic operon undercontrol of the T7 promoter system while HetI was expressedconstitutively on a separate plasmid. For biochemical assays, strainJK824 was typically grown in Luria Broth supplemented with 10% glycerolat 30° C. to an O.D. (600 nm) of ˜1 and then Isopropylβ-D-1-thiogalactopyranoside (IPTG) was added (to a final concentrationof 1 mM) to induce the production of the T7 polymerase. Approximately 4hrs after induction, the cells were harvested, washed and resuspended inBuffer KE (Buffer K—50 mM NaPO₄, pH 6.8 with 10% glycerol—which alsocontained 1 mM EDTA and 2 mM dithioerytheitol). The cells were rupturedby passage through a French Pressure Cell (16,000 psi) and thehomogenate separated into aliquots and stored at −80° C. for use in thePUFA synthase activity enhancement assay.

In vitro PUFA synthase activity enhancement assays: The in vitro PUFAsynthase assay involves measurement of radioactivity, from ¹⁴C labeledmalonyl-CoA, incorporated into DHA and DPAn-6. The generalcharacteristics of a PUFA synthase assay are described in Metz et al.,2009, Plant Physiology and Biochemistry 47:472-478. The typicalenhancement assay had a final volume of 100 μL—consisting of 40 μL ofthe homogenate derived from E. coli strain JK824 (described above), 40uL of a sample to be evaluated (e.g., homogenates of Schizochytriumstrains, or chromatographic fractions) and 10 μL of reaction cocktail(containing a mixture of cold and [2-¹⁴C]-malonyl-CoA and NADPH). TheJK824 homogenate and the sample to be evaluated were mixed just prior toaddition of the reaction cocktail. The components of the reactioncocktail were adjusted so that the final concentrations of malonyl-CoAand NADPH in the assay were 50 μM and 2 mM, respectively. Buffer Kserved as the negative control for the enhancement of activity and wasalso used to adjust the volume of the samples being evaluated to 40 μLwhen less than that amount was being tested. The assay reactions werecarried out in glass tubes in a room temperature (˜21° C.) water bath.The time of incubation was dependent on the experimental requirements.

The in vitro assay products of the Schizochytrium 20888 PUFA synthaseare free fatty acids (Metz et al., 2009, Plant Physiology andBiochemistry 47:472-478). The incorporation of radioactivity into thesefree fatty acids was routinely measured by extraction with organicsolvents and lipid class separation on TLC plates. Occasionally theextracted lipids were converted to FAMEs prior to separation on TLCplates to confirm that DHA and DPAn-6 were the primary fatty acids beinglabeled in the assay. The in vitro assay reactions were stopped by oneof two methods depending on the work-up protocol. For conversion offatty acids to fatty acid methyl-esters (FAMEs) using an acidic method,the reaction was stopped by adding the FAME reagent (see below). Forextraction of lipids without derivatization, the reaction was stopped byaddition of 125 μL of isopropanol:acetic acid (4:1 v/v) (see below).

Acidic FAME Protocol:

the reaction was stopped by adding 2.0 mL of 4% HCl in methanol plus 50μL toluene, the glass tubes were sealed with Teflon lined caps andheated at 100° C. for 1 hr. The reaction was cooled to room temperature,then 1.0 mL of hexane and 0.5 mL water was added, further vortexed thenleft to separate. If desired, a portion can be removed for liquidscintillation counting (LSC). An aliquot of ˜600 μL of organic phase wastransferred to a new tube and the solvent was removed under N₂. Theresidue was dissolve in 50 μL hexane and spotted onto either Silica gel60 A TLC plates (develop with hexane:diethyl-ether:acetic acid—70:30:2)or Silica Gel G plates soaked in 10% AgNO₃/90% acetonitrile (activatedfor 30 min at 100° C. prior to use) (developedw/hexane:diethyl-ether/acetic acid—70:20:2). The plates were left to airdry and radioactive areas were detected using phosphorimagingtechnology.

HIP Protocol—Extraction of Underivatized Lipids:

As indicated above, the reaction was stopped by adding 125 μL ofisopropanol:acetic acid (4:1 v/v) then adding 2 mL of hexane:isopropanol(3:2, v/v), further vortexed then 1 mL of 6.7% (w/v) sodium sulfate wasadded and vortexed again. The phases were left to separate. If desired,a portion of the organic (upper) phase was transferred for LSC then therest (˜1.0 mL) was transferred to a new tube. The solvent was removewith N₂ gas and the residue was dissolved in 50 μL of hexane. The samplewas spotted on a silica gel 60 A TLC plate and developed withhexane:diethyl-ether:acetic acid (70:30:2). The plates were let air dryand radioactive areas were detected using phosphorimaging technology.

Assay for identifying PUFA synthase enhancing factor proteins: for thePUFA synthase enhancement assay, extracts derived from one of severalSchizochytrium strains were combined with the above mentioned E. coliextract prior to addition of the ¹⁴C labeled malonyl-CoA and NADPH.Reactions in which the extraction buffer solution was combined with theE. coli material instead of the cell extracts served as negativecontrols. Several strains were used for these experiments.

Example 2

The following example describes the strains, derived from Schizochytrium20888, that were utilized to develop the in vitro PUFA synthaseenhancement assay and to identify the enhancing factors.

Quad K-O: Quad-KO is a Schizochytrium 20888 strain in which the threePUFA synthase subunit open reading frames (Orfs) and the FAS Orf weredeleted and replaced with antibiotic resistance gene cassettes. Thisstrain was created using the methods described in Hauvermale et al.,2008, Lipids, 41: 739-747, Metz et al., 2009, Plant Physiology andBiochemistry 47:472-478, and Roessler et al., 2006, U.S. Pat. No.7,001,772. The Quad-KO strain requires supplementation of the media withboth saturated and long chain polyunsaturated fatty acids for growth.Homogenates of this strain do not have the ability to incorporateradioactivity from ¹⁴C malonyl-CoA into fatty acids that are readilyextracted by organic solvents. Extracts from this strain were used toestablish the initial parameters for chromatographic enrichment offactors responsible for enhancement of PUFA synthase activity in the invitro assays.

AC66: The cell wall of Schizochytrium 20888 is difficult to disrupt thusmaking preparation of cell-free homogenates challenging. AC66 is astrain of Schizochytrium 20888 that was selected after chemicalmutagenesis based on colony morphology and was subsequently found to beeasily disrupted by sonication. The fatty acid profile and oilaccumulation properties of strain AC66 are very similar to those of itsparent. The ease of cell disruption by sonication greatly simplifiespreparation of cell-free homogenates for biochemical work.

AC66/DGAT KO: AC66/DGAT KO is a strain derived from AC66 in which a geneencoding a particular diacyl glycerol acyltransferase in Schizochytrium20888 has been inactivated (described in Metz et al., 2013, US PatentApplication Publication No. 2013-0150599, referred to in that patent asDAGAT-1). This strain, while prototrophic, does not accumulatesignificant amounts of oil. The lack of oil simplifies biochemicalanalysis and the initial steps of chromatographic enrichments of thePUFA synthase enhancing factors.

Example 3

The following example describes a procedure for chromatographicenrichment of the PUFA synthase in vitro activity enhancing factor(s)(EF-X) from extracts derived from strains of Schizochytrium 20888. Itwas determined subsequently by the experiments described in Example 4that EF-X from extracts derived from strains of Schizochytrium 20888actually contains two enhancing factors: Sz-TE2 and Sz-TE3.

Several chromatographic matrixes were tested for utility in enrichmentof the EF-X. Initial testing was done using extracts from the Quad-KOstrain since it lacks the FAS and PUFA synthase activities that couldconfound the enhancement assay. It was subsequently discovered that, byuse of appropriate conditions, the EF-X activity present in cell freeextracts of Schizochytrium 20888 could be cleanly separated from boththe FAS and PUFA synthase activities by use of a Mimetic Blue A SAcolumn. The starting material used for the example described below wasderived from the AC66/DGAT-KO strain of Schizochytrium 20888.

Cell free homogenate preparation: Cells of the AC66/DGAT-KO strain weregrown to mid-log phase, harvested by centrifugation, resuspended in‘Buffer K’ (50 mM NaPO₄, pH 6.8 with 10% glycerol) and lysed bysonication. The homogenate was centrifuged (100,000×g×1 hr) and thesupernatant collected and passed through a 0.2 μM filter to yield thestarting material (S100F) used for chromatography.

Mimetic Blue SA chromatography: A Mimetic Blue SA matrix (Blue SA) wasused for the initial chromatographic step. Approximately 50 mL of S100Fwas loaded onto a 10 mL column which had been equilibrated with BufferK. The column was washed with Buffer K and then with Buffer K containing0.24 M NaCl. Bound EF-X activity was eluted by a linear salt gradient(from 0.24 M to 2 M NaCl over 40 mL). Fractions (5 mL) were collectedand assayed. Those with the highest EF-X activity (in the middle portionof the gradient) were pooled and taken to the next step.

Hydrophobic Interaction Chromatography (HIC): A portion (10 mL) of thepooled material from the Blue SA column was loaded directly on to a 1 mLHiTrap Phenyl-FF cartridge (GE HealthCare) which had been equilibratedwith Buffer K containing 2 M NaCl. After washing with equilibrationbuffer, the bound EF-X activity was eluted using a 10 column volumereverse salt gradient (2 M to 0 M NaCl in Buffer K). Active fractionsfrom multiple runs were pooled for the next step. The column was cleanedwith 0.5 M NaOH between the runs.

Cation exchange chromatography: Pooled fractions from the HIC step wereconcentrated and desalted using ultrafiltration spin columns. A portion(2 mL) of the desalted material was loaded onto a 1 mL UNO-S cationexchange column (Bio-Rad) which had been equilibrated with Buffer K.After washing with Buffer K, the bound EF-X activity was eluted using a10 column volume salt gradient (0 M to 1 M NaCl in Buffer K). Fractions(0.5 mL) were collected and EF-X activity assayed.

Size exclusion chromatography: Two fractions from the cation exchangecolumn with the highest EF-X activity were combined then concentratedand desalted using ultrafiltration spin columns. A portion (500 μL) ofthis material was loaded on to a SuperDex 200 column (GE Health Care)which had been equilibrated with Buffer K. The EF-X activity wasretained by the matrix and eluted as a single peak with an apparentmolecular mass, estimated by comparison to protein standards of from 40to 80 kDa. SDS-PAGE analysis (with silver staining of the gel) revealedonly a few distinct polypeptide bands in the fractions with EF-Xactivity—and only three bands whose staining intensity appeared tocorrelate with the activity detected in those same fractions. Themolecular masses of these proteins were estimated to be ˜80 kDa, ˜37 kDaand ˜17 kDa, by comparison to protein standards separated on the samegel. Portions of the relevant fractions from the SuperDex 200 columnwere used for peptide generation and sequencing procedures. Peptidesequencing in conjunction with a Schizochytrium 20888 genome databasewas performed and EF-X candidates were identified.

Example 4

The following example describes the procedure utilized to identify twocandidate proteins for association with the enhancement of in vitro PUFAsynthase activity and the molecular characterization of thosecandidates.

LC-MSMS (liquid chromatography-tandem mass spectrometry) analysis wasperformed on tryptic peptides generated from fractions derived from thefinal chromatographic step (SuperDex 200 column separation) of Example2. Candidate proteins for association with the Schizochytrium 20888 EF-Xwere identified. The method involves LC-MSMS sequencing (with aSchizochytrium 20888 genome sequence derived predicted protein databaseas a reference) in combination with correlation of relative peptideabundance determined by spectral counting of the LC-MSMS data with theEF-X activity assay results for each fraction. A list of 17 candidateproteins was generated. The top two candidates were identified as likelythioesterases by BLAST analysis. Both candidates showed homology to4-hydroxybenzoyl-CoA-like thioesterases. The predicted nucleotidesequences and the deduced amino acid translations of the two candidateOrfs are provided in the ‘Sequence Listing’ and described below. Thecandidate proteins were termed Sz-TE2 and Sz-TE3. The Sz-TE2 protein islikely to be associated with the ˜17 kDa band identified in the silverstained gel mentioned in the previous example while Sz-TE3 is likely tobe associated with the ˜37 kDa band. The third highest hit in theSpectral Counting list appeared to be correctly annotated in the genomedatabase and showed very high homology to a bi-functional enzyme of thefatty acid degradation pathway. This protein is likely to be associatedwith the ˜80 kDa identified in the silver stained gel. Follow up workwas carried out with the Sz-TE2 and Sz-TE3 candidates. The molecularcharacteristics of the open reading frames and predicted encodedproteins are described here:

Sz-TE2: The nucleotide sequence of the predicted Sz-TE2 Orf contained411 bps (without the stop codon) and the translation encoded a 15.6 kDaprotein with 137 amino acids. A BLAST analysis of the nucleotidesequence against a proprietary Schizochytrium 20888 EST databaseidentified three matches that confirmed the entire Orf from the ATG to aTGA stop codon. The Orf nucleotide sequence is show in SEQ ID NO:5 andthe translation is shown in SEQ ID NO:1. As indicated, BLAST analysis ofthe Sz-TE2 protein sequence reveals homology to4-hydroxybenzoyl-CoA-like thioesterases.

Sz-TE3: The Sz-TE3 Orf annotated in the proprietary genome sequencedatabase was predicted to encode 881 amino acids and the coding regionto contain 3 introns within the overall length of the gene (start tostop) of 6,800 bp. A query of the proprietary Schizochytrium 20888 ESTdatabase with that entire sequence revealed that only the 3′ portion hadmatches. The eight ESTs identified in the BLAST search formed a contigthat included an Orf (from ATG to a TGA stop) whose sequence matchedthat of the genomic data. The Orf for the region confirmed by the ESTscontained 963 nucleotides (without the stop codon) which encoded a 36.6kDa protein with 321 amino acids. The Sz-TE3 Orf nucleotide sequence isshow in SEQ ID NO:6 and the translation is shown in SEQ ID NO:2. BLASTanalysis of the Sz-TE3 protein sequence also reveals homology to4-hydroxybenzoyl-CoA-like thioesterases—but in this case there are twosimilar regions: i.e., two adjacent regions, both with homology to the4-hydroxybenzoyl-CoA-like thioesterases.

Example 5

The following example describes the verification that candidateproteins, Sz-TE2 and Sz-TE3, are indeed associated with enhancement ofin vitro Schizochytrium 20888 PUFA synthase activity and suggest thatthey have optimal activity when expressed together.

Construction of E. coli expression plasmids containing Sz-TE2 andSz-TE3: The Orfs encoding Sz-TE2 and Sz-TE3 were cloned separately andtogether into two Novagen Duet vectors: pETDuet™ (carrying the Ampresistance marker) and pCOLADuet™ (carrying the Kan resistance marker).In both cases, the Sz-TE2 Orf was cloned into the MCS-2 and the Sz-TE3was cloned into the MCS-1. The final nucleotide sequences Orfs for bothOrfs in the constructs were identical to the native sequences. TheNovagen Duet plasmids utilize the T7 expression system and require hostcell lines containing an inducible T7 polymerase, e.g., BLR(DE3) orBL21(DE3). IPTG (0.5 mM) was used to induce expression of the genes.

Expression of Sz-TE2 and Sz-TE3 in E. coli—testing for proteinsolubility: A test of solubility of the Sz-TE2 and Sz-TE3 proteins in E.coli was carried out by expressing the proteins, either separately ortogether, collecting the induced cells and using the Novagen“BugBuster®” reagent and centrifugation protocol to separate ‘soluble’proteins from cell debris and non-soluble proteins. Samples of the wholecells and the soluble and non-soluble fractions were treated with SDSand analyzed by SDS-PAGE. A Coomassie Blue stained gel in which theproteins present in whole cells and the ‘soluble’ and ‘non-soluble’fractions from the several E. coli strains had been separated wasexamined. This revealed that a well stained protein band associated withSz-TE2 was present in the soluble fractions both when expressed byitself and when co-expressed with Sz-TE3. In contrast, when Sz-TE3 wasexpressed by itself, very little protein was detected in the solublefraction while a prominent band was present in the non-soluble fraction.When Sz-TE3 was co-expressed with Sz-TE2, the majority of the Sz-TE3protein was now detected in the soluble fraction. These data suggest aninteraction between Sz-TE3 and Sz-TE2 which increases the solubility ofSz-TE3. They also indicate that determination of the activity of Sz-TE3when expressed by itself in this particular E. coli system may becompromised by its lack of solubility.

Use of E. coli expressed Sz-TE2 and Sz-TE3 in the PUFA synthase activityenhancement assay: The Orfs encoding Schizochytrium 20888 EF-Xcandidates Sz-TE2 and Sz-TE3 were cloned separately and together intoNovagen Duet vectors as described above. E. coli strains containing theTE constructs were grown in LB medium at 32° C. Expression was inducedby addition of IPTG and the incubation continued for 3 to 5 hours. Cellswere collected by centrifugation and resuspended in Buffer K (50 mMNaPO₄ pH 6.8, 10% glycerol) and lysed using a French Pressure cell(16,000 psi—two passes). Aliquots of the homogenates were either storeddirectly, or centrifuged (20,000×g×20 min) to yield a supernatantfraction (S20) before being aliquoted and stored at −80° C. Thehomogenates and supernatant fractions were diluted 6× in Buffer K priorto being combined with the separately prepared homogenate of JK824(expressing the Schizochytrium 20888 PUFA synthase genes and HetI) forthe enhancement assay. In some assays, equal volumes of extracts fromstrains expressing Sz-TE2 or Sz-TE3 were combined—if tested alone,Buffer K was used to equalize the volumes. The reactions are started byaddition of ¹⁴C-malonyl CoA plus NADPH and run for 20 to 30 min at roomtemperature (˜21° C.) then stopped by addition of isopropanol/aceticacid. Neutral lipids were extracted using a hexane/isopropanol solutionand separated by normal phase TLC (Metz et al., 2009, Plant Physiologyand Biochemistry, 47:472-478). FIG. 1 shows the results of the activityassays when the extracts (homogenates or supernatant fractions) fromthese strains, or Buffer K, were combined with extracts from JK824. 2Hand 3H refer to Sz-TE2 and Sz-TE3, respectively, in homogenatesfractions. 2S and 3S refer to Sz-TE2 and Sz-TE3, respectively, insupernatant fractions. The data in the figure indicate that while theaddition of Sz-TE2 or Sz-TE3 alone did not enhance the PUFA synthaseactivity, significant enhancement (6 to 9 fold) was obtained when theywere combined. Additionally, the enhancing effect was observed with boththe homogenate and the supernatant fractions. These results confirm thatthe candidates, Sz-TE2 and Sz-TE3, are indeed associated with the EF-Xactivity and that both are likely to be required for that activity.

Example 6

The following example describes the effects of inactivation of theSz-TE2 or Sz-TE3 genes in the Quad-KO strain of Schizochytrium 20888 onthe EF-X activity in extracts derived from those strains. The resultsindicate that the Sz-TE2 and Sz-TE3 genes encode the primary factorsassociated with the in vitro PUFA synthase enhancing activity inextracts of Schizochytrium 20888.

Construction of Sz-TE2 and Sz-TE3 KO plasmid cassettes: The in vitroPUFA synthase enhancing activity was originally characterized bycombining extracts from E. coli with extracts prepared from the Quad-KOstrain of Schizochytrium 20888. In this strain, the genes encoding theFAS protein and the three subunits of the PUFA synthase were inactivatedby replacement of the coding regions with antibiotic resistancecassettes. For inactivation of the SzTE genes in the Quad-KO strain,plasmids were constructed in which the coding regions of either theSz-TE2 or Sz-TE3 were replaced with another antibiotic resistancecassette (paromomycin). Transformation of the Quad-KO strain was carriedout using the Biolistic system (Roessler et al., 2006 U.S. Pat. No.7,001,772) and paromomycin resistant colonies were selected on platessupplemented with both short chain saturated fatty acids and with DHA.The structures of the Sz-TE2 and Sz-TE3 loci in the putative KO strainswere verified by sequencing of cloned PCR fragments generated by usingprimers targeted to regions outside of the DNA flanking used in thetransformation cassettes. Two independent transformants for each of theSz-TE2 and Sz-TE3 KOs were grown in liquid medium, the cells collectedby centrifugation and resuspended in Buffer K. Cell homogenates wereprepared by shaking with glass beads which were then separated from thehomogenates by filtration. The homogenates were then centrifuged(20,000×g×10 min) to yield a fraction (S20) that was used in the invitro PUFA synthase activity enhancement assay. FIG. 2 shows the resultsof the those assays along with the activity of the E. coli extractitself (combined with Buffer K) and a sample in which a S20 fractionfrom the parent Quad-KO strain has been added. The addition of theQuad-KO extract resulted in an ˜8-fold increase in PUFA synthaseactivity in the assay (a typical enhancement of activity observed withthis type of extract). Use of the extracts from strains in which eitherthe Sz-TE2 or the Sz-TE3 gene have been disrupted resulted in only aminor increase in the E. coli expressed PUFA synthase activity. Thesedata demonstrate that Sz-TE2 and Sz-TE3 are responsible for the majorityof the enhancing activities observed in extracts of Schizochytrium 20888cells and that expression of both Sz-TE2 and Sz-TE3 genes are needed forthe appearance of the enhancing activity.

Example 7

The following example describes the effects of inactivation of Sz-TE2and Sz-TE3 and both genes in Schizochytrium 20888. The results revealthe in vivo roles of Sz-TE2 and Sz-TE3: although DHA and DPAn-6 stillaccumulate in the KO strain, their levels are significantly reduced.

The Sz-TE2 and Sz-TE3 proteins were identified using an in vitro PUFAsynthase enhancement assay. The potential role of these proteins in PUFAproduction and accumulation in cells of Schizochytrium 20888 wasinvestigated by inactivation of the corresponding genes in the wild typeSchizochytrium 20888 background. Sz-TE2 and Sz-TE3 KO constructs weremade using the methods described above. For this experiment, the Sz-TE2coding region was replaced with a paromomycin resistance cassette andthe Sz-TE3 with a Zeocin resistance cassette. Sz-TE2 and Sz-TE3 wereinactivated separately in that background to generate Sz-TE2 and Sz-TE3KO strains. Double Sz-TE KO strains were then created by transformationwith the second KO construct. Antibiotic resistant transformants thatgrew on plates that were not supplemented with DHA were obtained forboth the single and double KO genes. Insertional replacements of the TEgenes with the antibiotic resistance cassettes for several of the KOswere confirmed by cloning and sequencing of PCR product using primerstargeted to DNA regions located outside of the flanking DNA used for theKO constructs. Cells of the wild type strain as well as examples of thesingle and double TE KO strains were grown, collected and their fattyacid profiles determined Table I-A shows typical fatty acid profiles,shown as the % of total FAMEs, obtained for these strains while TableI-B shows those profiles expressed as the mg of the individual FAMEs pergram of dried biomass. It can be seen that all three versions of theSz-TE KO strains (Sz-TE2 KO, Sz-TE3 KO and the double KO) have fairlysimilar fatty acid profiles. Although all of the strains accumulate DHA,the amounts of that fatty acid have been significantly reduced relativeto the wild type. In the example shown, DHA decreased from ˜40% of totalFAME in the wild type to ˜34% in the TE2 KO and to ˜30% in the TE3 anddouble KOs. The higher amount of DHA detected in the Sz-TE2 KO strain,relative to those in which Sz-TE3 has been inactivated, suggests thatSz-TE3 may have some activity on its own. Additionally, the ratio of DHAto DPAn-6 in all of the strains has been lowered, from ˜2.3 in theparental strain to <2 in all of the TE KO strains. Although the DPAn-6level, as a % of total FAME, has not been significantly altered in theKO strains, the amount produced—when calculated as mg of DPAn-6 FAME pergram of biomass has been reduced. Although the reduction in DHA andDPAn-6 in the KO strains has been partially compensated by an increasein other fatty acids (derived from the FAS), the total mg FAME is stillreduced (Table I-B). These data indicate that while the products of thePUFA synthase (DHA and DPAn-6) still accumulate in Schizochytrium 20888strains in which the Sz-TE2 and or Sz-TE3 genes have been inactivated,the levels of those two PUFAs are higher when both genes are intact. Thedata provide a rationale for why DHA and DPAn-6 are observed toaccumulate when the Schizochytrium 20888 PUFA synthase (along with anappropriate PPTase) is expressed in heterologous organisms and suggestthat co-expression of Sz-TE2 plus Sz-TE3 may be a means to increase theaccumulation in those cells.

TABLE I-A X024-2 X024-4 20888 X022-1 X023-17 SzTE2 + SzTE2 + Fatty acidWT SzTE2 KO SzTE3 KO 3 KO 3 KO C14:0* 4.9 3.2 4.3 6.0 5.4 C16:0* 29.630.3 34.6 40.1 35.8 C16:1* 0.9 2.4 3.4 2.5 2.5 C18:0* 0.9 1.6 1.8 1.61.5 C18:1 N7 0.6 2.8 3.8 2.4 2.8 C20:5 N3* 1.0 1.5 1.1 0.9 0.9 C22:4 N90.7 1.4 1.3 1.0 1.1 C22:5 N6* 17.7 18.5 16.3 14.3 16.7 C22:6 N3* 40.434.3 29.8 27.9 29.2 DHA:DPAn6 2.3 1.9 1.8 1.9 1.7 DHA + DPAn6 58.1 52.846.1 42.2 45.8

Table I-A shows fatty acid profiles—as % of total FAME—of wild typeSchizochytrium 20888 and strains in which the following genes have beeninactivated by insertional mutagenesis: Sz-TE2 (X022-1) or Sz-TE3(X023-17) or both Sz-TE2 and Sz-TE3 (X024-2 and X024-4). Only thosefatty acids with levels>1% of the total FAMEs are shown.

TABLE I-B X024-2 X024-4 20888 X022-1 X023-17 SzTE2 + SzTE2 + Fatty AcidWT SzTE2 KO SzTE3 KO 3 KO 3 KO C14:0* 14.1 7.2 10.1 15.4 13.5 C16:0*85.7 67.3 81.3 103.0 88.8 C16:1* 2.5 5.3 7.9 6.5 6.1 C18:0* 2.6 3.6 4.24.2 3.8 C18:1 N7 1.7 6.2 9.0 6.2 7.0 C20:5 N3* 2.9 3.3 2.7 2.4 2.3 C22:4N9 2.0 3.2 3.0 2.6 2.6 C22:5 N6* 51.1 41.1 38.3 36.9 41.3 C22:6 N3*116.9 76.1 70.0 71.7 72.2 Sum FAME 289.4 222.0 235.3 257.0 247.8

Table I-B shows fatty acid profiles—mg FAME per gram of dried biomass—ofwild type Schizochytrium 20888 and strains in which the following geneshave been inactivated by insertional mutagenesis: Sz-TE2 (X022-1) orSz-TE3 (X023-17) or both Sz-TE2 and Sz-TE3 (X024-2 and X024-4). Onlythose fatty acids listed in Table I-A are shown.

Example 8

The following example describes the effect on fatty acid profiles ofco-expression of Sz-TE2 and Sz-TE3 in E. coli expressing theSchizochytrium 20888 PUFA synthase and HetI. The data indicate thataccumulation of the products of the PUFA synthase (DHA and DPAn-6) canbe increased by co-expression of Sz-TE2 plus Sz-TE3.

Expression of the Schizochytrium 20888 PUFA synthase subunits along withHetI in E. coli results in accumulation of DHA and DPAn-6 (seeHauvermale et al., 2008, Lipids, 41:739-747 for details). Additionally,it was determined that all of the PUFAs in those cells were in the freefatty acid form, as opposed to being esterified (e.g., as components ofphospholipids). This observation was consistent with the results of invitro activity assays of extracts from these cells; i.e., the productsof the Schizochytrium 20888 PUFA synthase reactions were detected asfree fatty acids. The ‘enhancement’ of the free fatty acid products ofthe in vitro PUFA synthase activity formed the basis of the assaydeveloped for identification of the EF-X factors. The fatty acidprofiles of E. coli strains in which the Sz-TE2 and Sz-TE3 proteins wereco-expressed, either separately or together, along with theSchizochytrium 20888 PUFA synthase system were also determined E. colistrain JK824 (expressing the Schizochytrium 20888 PUFA synthase subunitsand HetI) were transformed with the pCOLA™ duet vector containing eitherSz-TE2 (TE2) or Sz-TE3 (TE3) or both Sz-TE2+Sz-TE3 (TE2+TE3). Verifiedtransformants were grown at 32° C. in 765 medium supplemented with 10%(wt/vol) glycerol (Hauvermale et al., 2008, Lipids, 41:739-747). Thecells were harvested 20 hrs after induction with IPTG, washed with 50 mMTris pH 7.5, freeze dried and the fatty acids converted to FAMEs andanalyzed by GC. The results are shown in the following Tables.

TABLE II-A TE2 TE2 TE3 TE3 TE2 + 3 TE2 + 3 Fatty Acid JK824 JK1350JK1351 JK1346 JK1347 JK1349 JK1348 C12:0* 5.6 5.4 5.4 5.0 5.8 4.5 5.2C14:0* 3.6 3.8 4.1 3.3 4.2 3.1 3.8 C16:0* 17.8 18.2 20.5 17.1 19.5 15.617.5 C16:1* 8.1 9.4 6.2 7.5 7.3 7.5 6.2 Unknown 1 6.0 6.1 6.9 6.2 6.85.3 6.3 C18:1 N7 26.6 26.8 23.6 26.1 24.2 23.2 20.9 Unknown 3 5.1 4.37.3 6.6 5.2 5.0 5.3 Unknown 4 7.8 7.2 8.0 7.6 8.0 7.0 7.5 C22:5 N6* 4.65.0 3.4 4.9 4.0 6.4 5.7 C22:6 N3* 9.0 8.7 8.4 9.6 8.8 17.4 15.9 DHA +DPAn6 13.6 13.7 11.8 14.5 12.8 23.9 21.5 DHA/DPAn6 2.0 1.7 2.5 2.0 2.22.7 2.8

Table II-A shows fatty acid profiles—as % of total FAME—of E. colistrain JK824 (expressing the Schizochytrium 20888 PUFA synthase subunitsand HetI) and of strains expressing either Sz-TE2 (TE2) or Sz-TE3 (TE3)or both (TE2+3) in the JK824 background. Only those fatty acids withlevels>1% of the total FAMEs are shown.

TABLE II-B TE2 TE2 TE3 TE3 TE2 + 3 TE2 + 3 Fatty Acid JK824 JK1350JK1351 JK1346 JK1347 JK1349 JK1348 C12:0* 3.0 3.0 2.7 2.8 3.1 2.7 3.2C14:0* 1.9 2.1 2.1 1.9 2.3 1.9 2.3 C16:0* 9.5 10.0 10.4 9.6 10.6 9.410.7 C16:1* 4.3 5.2 3.1 4.2 3.9 4.5 3.8 Unknown 1 3.2 3.4 3.5 3.5 3.73.1 3.8 C18:1 N7 14.2 14.7 11.9 14.7 13.0 13.9 12.7 Unknown 3 2.7 2.43.7 3.7 2.8 3.0 3.2 Unknown 4 4.2 4.0 4.0 4.2 4.3 4.2 4.6 C22:5 N6* 2.52.8 1.7 2.8 2.2 3.9 3.5 C22:6 N3* 4.8 4.8 4.2 5.4 4.7 10.5 9.7 Sum FAME53.2 55.0 50.4 56.1 53.9 60.0 60.9 DHA + DPAn6 7.3 7.5 5.9 8.2 6.9 14.413.1

Table II-B shows fatty acid profiles—as mg of FAME/gram dried biomass—ofthe E. coli strains shown in Table II-A. Only those fatty acids includedin Table II-A are shown.

The data in Table II-A indicate that co-expression of either Sz-TE2 orSz-TE3 alone with the Schizochytrium 20888 PUFA synthase system in E.coli does not significantly alter the fatty acid profiles (as % of totalFAME) relative to the parental strain (JK824). In contrast, expressionof Sz-TE2 and Sz-TE3 together results in an approximately two-foldincrease in the amount of DHA in those cells. Interestingly, there isalso an increase in the amount of DPAn-6, but it is less than for DHA,resulting in an increase in the ratio of DHA to DPAn-6—from ˜2.0:1 to˜2.7:1. This higher ratio is closer to what is typically observed in oilfrom Schizochytrium 20888 itself

The data shown in Table II-B indicate that the increase in % PUFAobserved when Sz-TE2 and Sz-TE3 are both expressed is accompanied by anincrease in accumulation of the total mg of FAME per gram of biomass.Furthermore that increase is specifically associated with an increase inmg of DHA and DPAn-6 while the amounts of other fatty acids arerelatively unchanged. Previous analysis indicated that the products ofthe PUFA synthase accumulate as free fatty acids in E. coli, and it islikely that additional PUFAs in these cells are also present as freefatty acids rather than being incorporated into cellular membranes.

Example 9

The following example describes the effect on fatty acid profiles ofco-expression of Sz-TE2 and Sz-TE3 in yeast expressing theSchizochytrium 20888 PUFA synthase and HetI. The data indicate thataccumulation of the products of the Schizochytrium 20888 PUFA synthase(DHA and DPAn-6) can be increased by co-expression of Sz-TE2 plusSz-TE3. They also indicate that Sz-TE3 has some activity when expressedwithout Sz-TE2.

Cloning of Sz-TE2 and Sz-TE3 Orfs into yeast expression vectors:Expression of the Schizochytrium 20888 PUFA synthase (PFA 1, 2 and 3)along with HetI in yeast results in accumulation of DHA and DPAn-6 inthose cells (Metz et al., US Patent Application Publication No.2013-0150599). One of the strains created for expressing this systemcontained the genes in the following vectors: PFA1 in a pYES-Leu vector(the Orf codons were modified for yeast expression), PFA2 in apYES3-Tryp vector (the Orf codons were modified for yeast expression)and PFA3 and HetI in a pESC-Ura vector (the native Orf sequences wereused for both genes). To test the effects of co-expression of theenhancing factor candidates with the Schizochytrium 20888 PUFAsynthase—Sz-TE2 and Sz-TE3 coding regions were cloned into a pESC-Hisvector, either separately or together. Sz-TE2 was cloned behind the Gal1 promoter (as a BamHI-XhoI fragment) while Sz-TE3 was cloned behind theGal 10 promoter (as a EcoRI-NotI fragment). Both DNA fragments retainedthe native Orf nucleotide sequences.

FAME profiles of yeast strains expressing the Schizochytrium 20888 PUFAsynthase system and Sz-TE2 and Sz-TE3: Yeast strains expressing eitherthe active Schizochytrium 20888 PUFA synthase (subunit genes plus HetI)alone or also containing either Sz-TE2 or Sz-TE3, alone or together,were grown on appropriate media. Expression of the transgenes wasinduced by resuspension of washed cells in media containing galactose.The cells were grown for 20 hrs at 30° C. after induction then collectedby centrifugation, freeze dried and their fatty acids converted to FAMEsusing acidic methanol and analyzed by GC. The FAME profiles of thevarious strains are shown in Table III (as % of total FAME). The controlstrain (with just the PUFA synthase genes plus HetI) produced DHA andDPAn-6 at levels consistent with previous observations (Metz et al., USPatent Application Publication No. 2013-0150599), i.e., 2.9% DHA and 1.8or 1.9% DPAn-6. Co-expression of Sz-TE2 alone did not increase the PUFAlevels. Co-expression of Sz-TE3 alone resulted in an ˜1.5× increase inPUFA accumulation but did not alter the DHA to DPAn-6 ratio.Co-expression of Sz-TE2 and Sz-TE3 together resulted in an ˜1.6×increase in PUFA accumulation with an ˜1.9× increase in DHA level.Additionally, the DHA to DPAn-6 ratio in the cells co-expressing theSz-TE2 plus Sz-TE3 increased from ˜1.5 to ˜2.7. As in the case ofexpression in E. coli, the Sz-TE2 by itself did not result in anincrease in PUFA accumulation. In contrast to the E. coli result, Sz-TE3by itself did increase the accumulation both DHA and DPAn-6 in yeast.The apparent lack of solubility of Sz-TE3 (when expressed withoutSz-TE2) in E. coli may account for the different result in obtainedyeast. it is apparent that the outcome can be affected by theheterologous host utilized for expression. The increase in theDHA:DPAn-6 ratio is again observed when Sz-TE2 and Sz-TE3 are bothexpressed (from ˜1.5 to ˜2.7).

TABLE III % FAME 24 hrs TE2 TE2 TE3 TE3 TE2 + 3 TE2 + 3 Fatty AcidBRY4.11-3 BRY4.11-4 YMR1-3 YMR1-4 YMR2-3 YMR2-4 YMR3-3 YMR3-4 C12:0* 1.71.6 2.1 2.3 1.8 1.9 2.1 2.1 C14:0* 1.4 1.3 1.7 1.8 1.3 1.3 1.6 1.6C14:1* 0.4 0.4 0.5 0.6 0.4 0.4 0.4 0.5 C16:0* 17.5 17.1 19.0 18.9 18.318.5 18.5 18.3 C16:1* 41.5 41.4 43.5 45.7 39.7 40.4 40.8 41.8 C18:0* 6.16.1 5.8 5.2 6.1 6.0 5.6 5.2 C18:1 N9* 25.1 25.5 22.8 21.9 23.2 22.9 21.921.4 C18:1 N7* 1.0 1.0 0.9 0.8 0.9 0.8 0.7 0.7 C22:5 N6* 1.9 1.8 1.2 0.92.6 2.7 2.0 2.1 C22:6 N3* 2.9 2.9 1.9 1.6 4.8 4.1 5.5 5.6 DHA + DPAn64.7 4.7 3.1 2.5 7.4 6.8 7.5 7.7 DHA/DPAn6 1.5 1.6 1.6 1.8 1.8 1.5 2.72.7

Table III shows fatty acid profiles—as % of total FAME—of control yeaststrains (BRY4.11—expressing the Schizochytrium 20888 PUFA synthase, PFA1, 2, and 3, and HetI) and strains expressing either Sz-TE2 or Sz-TE3 orboth in the parental (BRY4.11) background. Only those fatty acids withlevels>1% of the total FAMEs are shown.

Example 10

This example demonstrates that homologs of Sz-TE2 and Sz-TE3 can bereadily identified in another Thraustochytrid that utilizes a PUFAsynthase.

Schizochytrium 9695 contains a PUFA synthase homologous to that found inSchizochytrium 20888 (Apt et al., U.S. Patent Application PublicationNo. 2010-0266564). A draft whole genome sequence of this organism wasgenerated and assembled into contigs. A translated BLAST (tblastn)search was carried out against a database set containing all of theassembled contigs using the Sz-TE2 and Sz-TE3 amino acid sequences asqueries. In both cases only 2 contigs were identified with significanthomology to the query sequences—and they were the same contigs in bothcases (in reverse order). The appropriate open reading frames wereidentified and the nucleotide sequences and their predicted translationsare listed. The amino acid homolog to Sz-TE2 has been designated B-TE2,the amino acid homolog to Sz-TE3 has been designated B-TE3. Thenucleotide sequences of these two Orfs along with the predictedtranslations are shown in the ‘Sequence Listing’ and are describedbelow.

The nucleotide sequence encoding B-TE2 and the predicted translation:The nucleotide sequence of the predicted Orf contained 468 bps (withoutthe stop codon) and the translation encoded a 17.36 kDa protein with 156amino acids.

The nucleotide sequence encoding B-TE3 and the predicted translation:The nucleotide sequence of the predicted Orf contained 963 bps (withoutthe stop codon) and the translation encoded a 36.5 kDa protein with 321amino acids.

Alignment of Sz-TE2 and Sz-TE3 amino acid sequences with the B-TEhomologs: FIG. 3A shows the alignment of Sz-TE2 with B-TE2. Theidentical residues in this alignment are shown in light grey and theconservative changes in darker grey, respectively. Using Sz-TE2 as thereference, 52 in 137 amino acids are identical (38.0%) and there are 78in 137 positives (identical plus conservative changes) (56.9%). TheB-TE2 sequence has an additional region rich in serines and glycines inthe middle of the protein (shown as a gap in the alignment).

FIG. 3B shows the alignment of Sz-TE3 with B-TE3. Both Sz-TE3 and B-TE3contain 321 amino acids. In this alignment, 155 in 321 amino acids areidentical (48.3%) and there are 192 in 321 positives (59.8%). As in thecase of B-TE2, the B-TE3 sequence has an additional region rich inserine and glycines in the middle of the protein (shown as a gap in thealignment).

Example 11

This example demonstrates that B-TE2 and B-TE3 can enhance the activityof the Schizochytrium 20888 PUFA synthase in an in vitro assay. Itsuggests that the EF-Xs may play a general role of enhancing PUFAsynthase activities.

Construction of E. coli expression plasmids containing B-TE2 and B-TE3Orfs: The same basic strategy used for cloning Sz-TE2 and Sz-TE3 wasemployed for the B-TE homologs. Orfs encoding B-TE2 and B-TE3 werecloned separately and together into the two Novagen Duet vectors:pETDuet™ (carrying the Amp resistance marker) and pCOLADuet™ (carryingthe Kan resistance marker). In both cases, the B-TE2 Orf was cloned intothe MCS-1 and B-TE3 was cloned into the MCS-2. The final Orfs for bothgenes in the constructs were identical to the native Orfs listed above.

Expression of B-TE2 and B-TE3 in E. coli—testing for protein solubility:The same strategy used for assessing the solubility of the Sz-TE2 andSz-TE3 proteins in E. coli was employed for the B-TE homologs. Cellscontaining plasmids with B-TE2 and B-TE3, either separately or together,were grown on LB medium at 32° C. Synthesis of the proteins was inducedby addition of IPTG (0.5 mM) and incubation was continued for 3 to 5hrs. The cells were collected by centrifugation and the Novagen“BugBuster®” reagent (and centrifugation protocol) was used to separatesoluble proteins from cell debris and non-soluble proteins (presumablysequestered in inclusion bodies). Samples of the whole cells and thesoluble and non-soluble fractions were treated with SDS and analyzed bySDS-PAGE. Bands associated with B-TE2 and B-TE3 were readily detected inthe whole cell extracts, with the B-TE3 being very highly expressed.Comparison of the proteins in the soluble and the non-soluble fractionsreveals that B-TE2, when expressed by itself, primarily partitions tothe soluble fraction. In contrast, B-TE3 remains primarily in theinsoluble fractions. When B-TE2 and B-TE3 are co-expressed in the samestrain, most of both proteins are in the insoluble fractions, but aminor amount of both can be detected in the soluble fractions. Theseresults are similar to what was observed for Sz-TE2 and Sz-TE3—but theinsolubility of B-TE3 is more pronounced. As in the case of Sz-TE2 andSz-TE3, these data suggest an interaction between B-TE2 and B-TE3proteins which may increase the solubility of B-TE3 in the E. colisystem.

In vitro activity enhancement assays: addition of separately expressedB-TE2 and B-TE3 to the Schizochytrium 20888 PUFA synthase system:Aliquots of the cell homogenates and supernatant fractions from the E.coli strains described above were tested in the PUFA synthase in vitroactivity assay. The extracts were mixed with a homogenate of E. colistrain JK824 (expressing the Schizochytrium 20888 Pfa 1, 2 and 3 plusHetI), and the assay run as described above. As for the assays of Sz-TE2and Sz-TE3, the homogenates and supernatant fractions were diluted 6× inBuffer K prior to being combined with the separately prepared homogenateof JK824 in the enhancement assay (see Example 5, above, for additionaldetails). FIG. 4 shows the results of these activity assays along withcontrol assays (addition of buffer alone and addition of E. coliexpressed Sz-TE2 and Sz-TE3). In FIG. 4, “B” refers to Schizochytrium9695. “Sz” refers to Schizochytrium 20888. “2” refers to TE2. “3” refersto TE3. “S” refers to supernatant fraction. “H” refers to homogenatefraction. The data in the figure show that B-TE2 and B-TE3, whenexpressed together in E. coli, can significantly enhance the in vitroactivity of the Schizochytrium 20888 PUFA synthase. This effect is mostevident when the homogenate was used in the assay—the supernatantfraction from that strain was much less active. Additionally, theSchizochytrium 20888 and Schizochytrium 9695 derived B-TEs can bemixed—i.e., the B-TE2 can substitute for Sz-TE2 and B-TE3 can substitutefor Sz-TE3. These data validate the selection of B-TE2 and B-TE3 asenhancing factor proteins.

While various embodiments of the present invention have been describedin detail, it is apparent that modifications and adaptations of thoseembodiments will occur to those skilled in the art. It is to beexpressly understood, however, that such modifications and adaptationsare within the scope of the present invention.

What is claimed is:
 1. A recombinant nucleic acid molecule comprising anucleic acid sequence encoding a polypeptide that is at least 90%identical to an amino acid sequence of SEQ ID NO:1 or SEQ ID NO:3. 2.The recombinant nucleic acid molecule of claim 1, wherein thepolypeptide is at least 95% identical to an amino acid sequence of SEQID NO:1 or SEQ ID NO:3.
 3. The recombinant nucleic acid molecule ofclaim 1, wherein the polypeptide enhances the enzymatic activity of aPUFA synthase.
 4. The recombinant nucleic acid molecule of claim 3,wherein said recombinant nucleic acid molecule comprises a nucleic acidsequence which is an enzymatically active fragment of SEQ ID NO:1 or SEQID NO:3.
 5. The recombinant nucleic acid molecule of claim 1, whereinthe nucleic acid sequence encodes a polypeptide having an amino acidsequence of SEQ ID NO:1 or SEQ ID NO:3.
 6. The recombinant nucleic acidmolecule of claim 1, wherein the nucleic acid sequence is SEQ ID NO:5 orSEQ ID NO:7.
 7. A recombinant nucleic acid molecule comprising a nucleicacid sequence encoding a polypeptide that is at least 90% identical toan amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4.
 8. The recombinantnucleic acid molecule of claim 7, wherein the polypeptide is at least95% identical to an amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4.9. The recombinant nucleic acid molecule of claim 7, wherein thepolypeptide enhances the enzymatic activity of a PUFA synthase.
 10. Therecombinant nucleic acid molecule of claim 9, wherein said recombinantnucleic acid molecule comprises a nucleic acid sequence which is anenzymatically active fragment of SEQ ID NO:2 or SEQ ID NO:4.
 11. Therecombinant nucleic acid molecule of claim 7, wherein the nucleic acidsequence encodes a polypeptide having an amino acid sequence of SEQ IDNO:2 or SEQ ID NO:4.
 12. The recombinant nucleic acid molecule of claim7, wherein the nucleic acid sequence is SEQ ID NO:6 or SEQ ID NO:8. 13.An isolated protein encoded by the nucleic acid molecule of any one ofclaims 1 to
 12. 14. A recombinant nucleic acid molecule, comprising thenucleic acid molecule according to any one of claims 1 to 12,operatively linked to an expression control sequence.
 15. A recombinanthost cell comprising the recombinant nucleic acid molecule of claimclaim
 14. 16. The recombinant host cell of claim 15, wherein the hostcell is a microorganism.
 17. A genetically modified microorganism,wherein the microorganism has been genetically modified to express therecombinant nucleic acid molecule of any one of claims 1 to
 12. 18. Agenetically modified microorganism, wherein the microorganism has beengenetically modified to express at least one recombinant nucleic acidmolecule of any one of claims 1 to 6 and at least one recombinantnucleic acid molecule of any one of claims 7 to
 12. 19. The geneticallymodified microorganism of any one of claims 17 to 18, wherein theorganism endogenously expresses a PUFA synthase system, aphosphopantetheinyl transferase (PPTase), and/or an acyl-CoA synthetase(ACS).
 20. The genetically modified microorganism of claim 19, whereinthe microorganism is a Thraustochytriales microorganism.
 21. Thegenetically modified microorganism of claim 20, wherein themicroorganism is a Schizochytrium.
 22. The genetically modifiedmicroorganism of claim 19, wherein the microorganism is a bacterium. 23.The genetically modified microorganism of any one of claims 17 to 18,wherein the organism has been further genetically modified toexogenously express a PUFA synthase system, a phosphopantetheinyltransferase (PPTase), and/or an acyl-CoA synthetase (ACS).
 24. Thegenetically modified microorganism of claim 23, wherein the PUFAsynthase system comprises at least one functional domain from a PUFAsynthase system from a Thraustochytriales microorganism.
 25. Thegenetically modified microorganism of claim 24, wherein the PUFAsynthase system comprises at least one functional domain from a PUFAsynthase system from a Schizochytrium.
 26. The genetically modifiedmicroorganism of claim 23, wherein the PUFA synthase comprises at leastone functional domain from a PUFA synthase from a microorganism selectedfrom the group consisting of Schizochytrium sp. American Type CultureCollection (ATCC) No. 20888, Schizochytrium sp. American Type CultureCollection (ATCC) No. PTA-9695, Thraustochytrium 23B American TypeCulture Collection (ATCC) No. 20892, and a mutant of any of saidmicroorganisms.
 27. The genetically modified microorganism of claim 23,wherein the PUFA synthase comprises at least one functional domain froma PUFA synthase from a marine bacterium.
 28. The genetically modifiedmicroorganism of claim 27, wherein the microorganism is a microalga, ayeast, or a bacterium.
 29. The genetically modified microorganism of anyone of claims 23 to 28, wherein one or more nucleic acid sequencesencoding the PUFA synthase has been optimized to improve the expressionof the PUFA synthase in the microorganism.
 30. The genetically modifiedmicroorganism of any one of claims 19 to 29, wherein the microorganismcomprises at least one polyunsaturated fatty acid (PUFA) selected fromthe group consisting of: DHA (C22:6, n-3), DPA (C22:5, n-6 or n-3), EPA(C20:5, n-3), ARA (C20:4, n-6), GLA (C18:3, n-6), and/or SDA (C18:4,n-3).
 31. The genetically modified microorganism of any one of claims 19to 29, wherein the genetically modified microorganism comprises DHA,DPAn-6 and/or EPA.
 32. The genetically modified microorganism of claim18, wherein the amount of DHA, DPAn-6 and/or EPA produced in saidgenetically modified microorganism is higher than that is produced inthe counterpart microorganism which none of the recombinant nucleic acidmolecule of claims 1 to 12 is expressed.
 33. The genetically modifiedmicroorganism of claim 18, wherein the ratio of DHA:DPAn-6 produced insaid genetically modified microorganism is higher than that produced inthe counterpart microorganism which none of the recombinant nucleic acidmolecule of claims 1 to 12 is expressed.
 34. The genetically modifiedmicroorganism of claim 32 or claim 33, wherein the microorganism is amicroalga, a yeast, or a bacterium.
 35. A genetically modifiedmicroorganism, wherein the microorganism has been genetically modifiedto delete or inactivate the nucleic acid molecule of any one of claims 1to 12 expressed by the microorganism.
 36. The genetically modifiedmicroorganism of claim 35, wherein the microorganism is aThraustochytriales microorganism.
 37. The genetically modifiedmicroorganism of claim 3, wherein the microorganism is a Schizochytrium.38. An oil obtained from the genetically modified microorganism of anyone of claims 17 to
 37. 39. A method to produce an oil comprising atleast one polyunsaturated fatty acid (PUFA), comprising growing themicroorganism of any one of claims 17 to
 37. 40. An oil produced by themethod of claim
 39. 41. The oil of claim 38 or 40, wherein the oilcontains at least one polyunsaturated fatty acid (PUFA) selected fromthe group consisting of: DHA (C22:6, n-3), DPA (C22:5, n-6 or n-3),and/or EPA (C20:5, n-3).
 42. A food product or feed product thatcontains an oil of any one of claims 38, 40 and 41, or the geneticallymodified microorganisms of any one of claims 17 to
 37. 43. Apharmaceutical product that contains an oil of any one of claims 38, 40and
 41. 44. A method to produce an oil comprising at least one PUFA,comprising recovering an oil from the genetically modified microorganismof any one of claims 17 to
 37. 45. A method to produce at least onepolyunsaturated fatty acid (PUFA), comprising growing the geneticallymodified microorganism of any one of claims 17 to
 37. 46. A method toproduce at least one polyunsaturated fatty acid (PUFA), comprisingobtaining or recovering the PUFA from the genetically modifiedmicroorganism of any one of claims 17 to
 37. 47. A method to provide asupplement or therapeutic product containing at least one PUFA to anindividual, comprising providing to the individual genetically modifiedmicroorganism or a part thereof of any one of claims 17 to 37, an oil ofclaims 38, 40 and 41, a food product of claim 42, or a pharmaceuticalproduct of claim
 43. 48. A process for transforming a microorganism toexpress PUFAs, comprising transforming an microorganism with nucleicacid molecules encoding a PUFA synthase, with a nucleic acid moleculeencoding a phosphopantetheinyl transferase (PPTase), with a nucleic acidmolecule encoding an acyl-CoA synthetase (ACS), and with at least onenucleic acid molecule according to any one of claims 1 to 12.